Method for administering a cytokine to the central nervous system and the lymphatic system

ABSTRACT

The present invention is directed to a method for delivering cytokines to the central nervous system and the lymphatic system by way of a tissue innervated by the trigeminal nerve and/or olfactory nerve. Cytokines include tumor necrosis factors, interleukins, interferons, particularly interferon-β and its muteins such as IFN-β ser17 . Such a method of delivery can be useful in the treatment of central nervous system disorders, brain disorders, proliferative, viral, and/or autoimmune disorders such as Sjogren&#39;s disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication No. 60/200,708, filed Dec. 9, 1999, herein incorporated byreference.

FIELD OF THE INVENTION

[0002] The present invention is directed to a method for deliveringcytokines to the central nervous system and by the lymphatic system byway of a tissue innervated by the trigeminal nerve and/or olfactorynerve. Cytokines include tumor necrosis factors, interleukins,interferons, particularly β-interferon and its muteins such asIFN-β_(ser17). Such a method of delivery can be useful in the treatmentof central nervous system and/or brain disorders.

BACKGROUND OF THE INVENTION

[0003] The central nervous system (CNS) includes several tissues andorgans, such as the brain, the brain stem, and the spinal cord. Each ofthese organs and tissues is made up of a variety of different types ofcells and subcellular structures, e.g., neurons, glial cells, dendrites,axons, myelin, and various membranes. The CNS is isolated from theexternal world by several membranes that both cushion and protect theseorgans, tissues, cells, and structures. For example, the membranes thatform the blood-brain barrier protect the brain from certain contents ofthe blood. The blood-cerebrospinal fluid barrier protects other portionsof the CNS from many chemicals and microbes.

[0004] Access to the CNS for some substances is provided by specializedactive transport systems or through passive diffusion through theprotective membrane into the CNS. Present methods for delivering desiredtherapeutic agents to the CNS are typically invasive. For example, apump implanted into the chest cavity (an intracerebroventricular pump)can effectively deliver a variety of useful compounds to the brain.However, implanting such a pump requires surgery, which can entail avariety of serious complications. Certain compounds (e.g., epiduralpainkillers) can be injected directly through the protective membraneinto the CNS. Such injection is, however, impractical for mostmedications. Better methods for administering desired agents to the CNS,brain, spinal cord, and lymphatic channels are needed.

SUMMARY OF THE INVENTION

[0005] The present invention relates to a method for transporting ordelivering a cytokine, such as an interferon, an interleukin, or a tumornecrosis factor, preferably interferon-β, to the central nervous systemof a subject. The method employs administration of the cytokine to atissue innervated by the trigeminal nerve and/or olfactory nerve.

[0006] In one embodiment, the method administers the cytokine throughthe mucosa or epithelium of the nasal cavity, tongue, mouth, skin, orconjunctiva. In another embodiment, the method includes administering acomposition of the cytokine to the nasal cavity, under the tongue, tothe skin, or to the conjunctiva of the subject. The cytokine can then beabsorbed through a mucosa or epithelium and transported to the centralnervous system of the mammal.

[0007] In another embodiment, the method includes administering thecytokine in a manner such that the cytokine is absorbed through thetissue and transported into the central nervous system of the mammal bya neural pathway and in an amount effective to provide a protective ortherapeutic effect on a cell of the central nervous system.

[0008] The present invention further relates to a method fortransporting or delivering a cytokine, such as an interferon, aninterleukin, or a tumor necrosis factor, preferably interferon-β, to thelymphatic system of a subject. The method employs administration of thecytokine to a tissue innervated by the trigeminal nerve and/or olfactorynerve.

[0009] In another embodiment, the method includes administering thecytokine in a manner such that the cytokine is absorbed through thetissue and transported into the central nervous system of the mammal bya neural pathway and in an amount effective to modulate an immune orinflammatory response.

[0010] In other embodiments, the method of administering a cytokine isused for the treatment and/or prevention of central nervous systemdisorders, brain disorders, proliferative, viral, and/or autoimmunedisorders.

[0011] The composition can be of any form suitable for administration bythese routes and can include a carrier that facilitates absorption ofthe cytokine, transport of the cytokine by a neural pathway, and/ortransport of the cytokine to the lymphatic system, CNS, brain, and/orspinal cord. Preferred compositions include one or more of a solubilityenhancing additive, a hydrophilic additive, an absorption promotingadditive, a cationic surfactant, a viscosity enhancing additive, or asustained release matrix or composition, a lipid-based carrier,preferably a micellar or liposomal composition, a bilayer destabilizingadditive, or a fusogenic additive. The composition can be formulated asa cosmetic for dermal delivery.

BRIEF DESCRIPTION OF FIGURES

[0012]FIG. 1 shows the level of Betaseron in the blood stream over timefollowing both intravenous administration (I.V.) and intranasaladministration (I.N.) in a rat.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Routes of Administration

[0014] The method of the invention administers the cytokine to tissueinnervated by the trigeminal and olfactory nerves. Such nerve systemscan provide a direct connection between the outside environment and thebrain, thus providing advantageous delivery of a cytokine to the CNS,including brain, brain stem, and/or spinal cord. Cytokines are unable tocross or inefficiently cross the blood-brain barrier from thebloodstream into the brain. The methods of the present invention allowfor the delivery of the cytokine by way of the olfactory and/ortrigeminal nerve rather than through the circulatory system. This methodof administration allows for the efficient delivery of a cytokine to theCNS, brain, or spinal cord.

[0015] The Olfactory Nerve

[0016] The method of the invention includes administration of a cytokineto tissue innervated by the olfactory nerve. Preferably, the cytokine isdelivered to the olfactory area in the upper third of the nasal cavityand particularly to the olfactory epithelium.

[0017] Fibers of the olfactory nerve are unmyelinated axons of olfactoryreceptor cells that are located in the superior one-third of the nasalmucosa. The olfactory receptor cells are bipolar neurons with swellingscovered by hair-like cilia that project into the nasal cavity. At theother end, axons from these cells collect into aggregates and enter thecranial cavity at the roof of the nose. Surrounded by a thin tube ofpia, the olfactory nerves cross the subarachnoid space containing CSFand enter the inferior aspects of the olfactory bulbs. Once the cytokineis dispensed into the nasal cavity, the cytokine can undergo transportthrough the nasal mucosa and into the olfactory bulb and interconnectedareas of the brain, such as the hippocampal formation, amygdaloidnuclei, nucleus basalis of Meynert, locus ceruleus, the brain stem, andthe like.

[0018] The Trigeminal Nerve

[0019] The method of the invention administers the cytokine to tissueinnervated by the trigeminal nerve. The trigeminal nerve innervatestissues of a mammal's (e.g., human) head including skin of the face andscalp, oral tissues, and tissues of and surrounding the eye. Thetrigeminal nerve has three major branches, the ophthalmic nerve, themaxillary nerve, and the mandibular nerve. The method of the inventioncan administer the cytokine to tissue innervated by one or more of thesebranches.

[0020] The Ophthalmic Nerve and its Branches

[0021] The method of the invention can administer the cytokine to tissueinnervated by the ophthalmic nerve branch of the trigeminal nerve. Theophthalmic nerve innervates tissues including superficial and deep partsof the superior region of the face, such as the eye, the lacrimal gland,the conjunctiva, and skin of the scalp, forehead, upper eyelid, andnose.

[0022] The ophthalmic nerve has three branches known as the nasociliarynerve, the frontal nerve, and the lacrimal nerve. The method of theinvention can administer the cytokine to tissue innervated by the one ormore of the branches of the ophthalmic nerve. The frontal nerve and itsbranches innervate tissues including the upper eyelid, the scalp,particularly the front of the scalp, and the forehead, particularly themiddle part of the forehead. The nasociliary nerve forms severalbranches including the long ciliary nerves, the ganglionic branches, theethmoidal nerves, and the infratrochlear nerve. The long ciliary nervesinnervate tissues including the eye. The posterior and anteriorethmoidal nerves innervate tissues including the ethmoidal sinus and theinferior two-thirds of the nasal cavity. The infratrochlear nerveinnervates tissues including the upper eyelid and the lacrimal sack. Thelacrimal nerve innervates tissues including the lacrimal gland, theconjunctiva, and the upper eyelid. Preferably, the present methodadministers the cytokine to the ethmoidal nerve.

[0023] The Maxillary Nerve and its Branches

[0024] The method of the invention can administer the cytokine to tissueinnervated by the maxillary nerve branch of the trigeminal nerve. Themaxillary nerve innervates tissues including the roots of several teethand facial skin, such as skin on the nose, the upper lip, the lowereyelid, over the cheekbone, over the temporal region. The maxillarynerve has branches including the infraorbital nerve, thezygomaticofacial nerve, the zygomaticotemporal nerve, the nasopalatinenerve, the greater palatine nerve, the posterior superior alveolarnerves, the middle superior alveolar nerve, and the interior superioralveolar nerve. The method of the invention can administer the cytokineto tissue innervated by the one or more of the branches of the maxillarynerve.

[0025] The infraorbital nerve innervates tissue including skin on thelateral aspect of the nose, upper lip, and lower eyelid. Thezygomaticofacial nerve innervates tissues including skin of the faceover the zygomatic bone (cheekbone). The zygomaticotemporal nerveinnervates tissue including the skin over the temporal region. Theposterior superior alveolar nerves innervate tissues including themaxillary sinus and the roots of the maxillary molar teeth. The middlesuperior alveolar nerve innervates tissues including the mucosa of themaxillary sinus, the roots of the maxillary premolar teeth, and themesiobuccal root of the first molar tooth. The anterior superioralveolar nerve innervates tissues including the maxillary sinus, thenasal septum, and the roots of the maxillary central and lateralincisors and canine teeth. The nasopalantine nerve innervates tissuesincluding the nasal septum. The greater palatine nerve innervatestissues including the lateral wall of the nasal cavity. Preferably, thepresent method administers the cytokine to the nasopalatine nerve and/orgreater palatine nerve.

[0026] The Mandibular Nerve and its Branches

[0027] The method of the invention can administer the cytokine to tissueinnervated by the mandibular nerve branch of the trigeminal nerve. Themandibular nerve innervates tissues including the teeth, the gums, thefloor of the oral cavity, the tongue, the cheek, the chin, the lowerlip, tissues in and around the ear, the muscles of mastication, and skinincluding the temporal region, the lateral part of the scalp, and mostof the lower part of the face.

[0028] The mandibular nerve has branches including the buccal nerve, theauriculotemporal nerve, the inferior alveolar nerve, and the lingualnerve. The method of the invention can administer the cytokine to one ormore of the branches of the mandibular nerve. The buccal nerveinnervates tissues including the cheek, particularly the skin of thecheek over the buccinator muscle and the mucous membrane lining thecheek, and the mandibular buccal gingiva (gum), in particular theposterior part of the buccal surface of the gingiva. Theauriculotemporal nerve innervates tissues including the auricle, theexternal acoustic meatus, the tympanic membrane (eardrum), and skin inthe temporal region, particularly the skin of the temple and the lateralpart of the scalp. The inferior alveolar nerve innervates tissuesincluding the mandibular teeth, in particular the incisor teeth, thegingiva adjacent the incisor teeth, the mucosa of the lower lip, theskin of the chin, the skin of the lower lip, and the labial mandibulargingivae. The lingual nerve innervates tissues including the tongue,particularly the anterior two-thirds of the tongue, the floor of themouth, and the gingivae of the mandibular teeth. Preferably, the methodof the invention administers the cytokine to one or more of the inferioralveolar nerve, the buccal nerve, and/or the lingual nerve.

[0029] Tissues Innervated by the Trigeminal Nerve

[0030] The method of the invention can administer the cytokine to any ofa variety of tissues innervated by the trigeminal nerve. For example,the method can administer the cytokine to skin, epithelium, or mucosa ofor around the face, the eye, the oral cavity, the nasal cavity, thesinus cavities, or the ear.

[0031] Preferably, the method of the invention administers the cytokineto skin innervated by the trigeminal nerve. For example, the presentmethod can administer the cytokine to skin of the face, scalp, ortemporal region. Suitable skin of the face includes skin of the chin;the upper lip, the lower lip; the forehead, particularly the middle partof the forehead; the nose, including the tip of the nose, the dorsum ofthe nose, and the lateral aspect of the nose; the cheek, particularlythe skin of the cheek over the buccinator muscle or skin over the cheekbone; skin around the eye, particularly the upper eyelid and the lowereyelid; or a combination thereof. Suitable skin of the scalp includesthe front of the scalp, scalp over the temporal region, the lateral partof the scalp, or a combination thereof. Suitable skin of the temporalregion includes the temple and scalp over the temporal region.

[0032] Preferably, the method of the invention administers the cytokineto mucosa or epithelium innervated by the trigeminal nerve. For example,the present method can administer the cytokine to mucosa or epitheliumof or surrounding the eye, such as mucosa or epithelium of the uppereyelid, the lower eyelid, the conjunctiva, the lacrimal system, or acombination thereof. The method of the invention can also administer thecytokine to mucosa or epithelium of the sinus cavities and/or nasalcavity, such as the inferior two-thirds of the nasal cavity and thenasal septum. The method of the invention can also administer thecytokine to mucosa or epithelium of the oral cavity, such as mucosa orepithelium of the tongue; particularly the anterior two-thirds of thetongue and under the tongue; the cheek; the lower lip; the upper lip;the floor of the oral cavity; the gingivae (gums), in particular thegingiva adjacent the incisor teeth, the labial mandibular gingivae, andthe gingivae of the mandibular teeth; or a combination thereof.Preferably, the method of the invention administers the cytokine tomucosa or epithelium of the nasal cavity. Other preferred regions ofmucosa or epithelium for administering the cytokine include the tongue,particularly sublingual mucosa or epithelium, the conjunctiva, thelacrimal system, particularly the palpebral portion of the lacrimalgland and the nasolacrimal ducts, the mucosa of the lower eyelid, themucosa of the cheek, or a combination thereof.

[0033] Preferably, the method of the invention administers the cytokineto nasal tissues innervated by the trigeminal nerve. For example, thepresent method can administer the cytokine to nasal tissues includingthe sinuses, the inferior two-thirds of the nasal cavity and the nasalseptum. Preferably, the nasal tissue for administering the cytokineincludes the inferior two-thirds of the nasal cavity and the nasalseptum.

[0034] Preferably, the method of the invention administers the cytokineto oral tissues innervated by the trigeminal nerve. For example, thepresent method can also administer the cytokine to oral tissues such asthe teeth, the gums, the floor of the oral cavity, the cheeks, the lips,the tongue, particularly the anterior two-thirds of the tongue, or acombination thereof. Suitable teeth include mandibular teeth, such asthe incisor teeth. Suitable portions of the teeth include the roots ofseveral teeth, such as the roots of the maxillary molar teeth, themaxillary premolar teeth, the maxillary central and lateral incisors,the canine teeth, and the mesiobuccal root of the first molar tooth, ora combination thereof. Suitable portions of the lips include the skinand mucosa of the upper and lower lips. Suitable gums include thegingiva adjacent the incisor teeth and the gingivae of the mandibularteeth, such as the labial mandibular gingivae, or a combination thereof.Suitable portions of the cheek include the skin of the cheek over thebuccinator muscle, the mucous membrane lining the cheek, and themandibular buccal gingiva (gum), in particular the posterior part of thebuccal surface of the gingiva, or a combination thereof. Preferred oraltissue for administering the cytokine includes the tongue, particularlysublingual mucosa or epithelium, the mucosa inside the lower lip, themucosa of the cheek, or a combination thereof.

[0035] Preferably, the method of the invention administers the cytokineto a tissue of or around the eye that is innervated by the trigeminalnerve. For example, the present method can administer the cytokine totissue including the eye, the conjunctiva, and the lacrimal glandincluding the lacrimal sack, the skin or mucosa of the upper or lowereyelid, or a combination thereof. Preferred tissue of or around the eyefor administering the cytokine includes the conjunctiva, the lachrimalsystem, the skin or mucosa of the eyelid, or a combination thereof.Cytokine that is administered conjunctivally but not absorbed throughthe conjunctival mucosa can drain through nasolachrimal ducts into thenose, where it can be transported to the CNS, brain, and/or spinal cordas though it had been intranasally administered.

[0036] Preferably, the method of the invention administers the cytokineto a tissue of or around the ear that is innervated by the trigeminalnerve. For example, the present method can administer the cytokine totissue including the auricle, the external acoustic meatus, the tympanicmembrane (eardrum), and the skin in the temporal region, particularlythe skin of the temple and the lateral part of the scalp, or acombination thereof. Preferred tissue of or around the ear foradministering the cytokine includes the skin of the temple.

[0037] Cytokines

[0038] Cytokines can be administered to the CNS, brain, and/or spinalcord according to the present invention. Cytokines that can beadministered by the method of the invention are cytokines that areimmunomodulators, such as interleukins (i.e., IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9 and IL-10), interferons, and tumor necrosisfactor (i.e., TNF-α and TNF-β), and that have activities directed atcells of the immune system. These cytokines are of interest astherapeutic cytokines, for example, for treatment of viral diseases andcontrol of cancer. It is believed that such cytokines have not beenobserved to have neurotrophic activity, or to have other direct,beneficial effects on neurons characteristic of nerve growth factor andlike compounds. Thus, it was not expected that such cytokines should betransported into the CNS, brain, and or spinal cord, particularly not bya neural pathway, or from tissues innervated by the olfactory and/ortrigeminal nerves.

[0039] A preferred cytokine for use in the practice of the invention aremembers of the interferon family. Interferons (IFNs) are a family ofmolecules encompassing over 20 different proteins and are members of thecytokine family that induce antiviral, antiproliferative, antitumor,and/or cytokine effects. IFNs are relatively small, species-specific,single chain polypeptides, which are produced in response to a varietyof inducers, such as mitogens, polypeptides, viruses, and the like. Inhumans, IFNs are produced in forms α, β, γ, ω, and τ. Syntheticinterferons are also known in the art. See, for example, 6,114,145,herein incorporated by reference. Upon secretion from mammalian cells,interferon molecules bind to a receptor on the surface of a target celland elicit a chain of events, which can alter the amount and activity ofprotein in the target cell. Such alterations can include, for example,changes in gene transcription or enzymatic activity. A preferredinterferon for use in the practice of the invention is interferon-β(IFN-β), interferon-α (IFN-α), and interferon-γ (IFN-γ).

[0040] Biologically active variants of cytokines are also encompassed bythe method of the present invention. Such variants should retain thebiological activity of the cytokine. For example, when the cytokine isan interferon, such as IFN-α, IFN-β, IFN-γ, the ability to bind theirrespective receptor sites will be retained. Such activity may bemeasured using standard bioassays. Representative assays detecting theability of the variant to interact with an interferon receptor type Ican be found in, for example, U.S. Pat. No. 5,766,864, hereinincororpated by reference. Preferably, the variant has at least the sameactivity as the native molecule. Alternatively, the biological activityof a variant of the cytokine of the invention can be assayed bymeasuring the ability of the variant to increase viral resistance in acell line using a standard viral reduction assay. See for example, U.S.Pat. No. 5,770,191, herein incorporated by reference. Other assays forbiological activity include, anti-proliferative assays as described inU.S. Pat. No. 5,690,925.

[0041] Suitable biologically active variants can be fragments,analogues, and derivatives of the cytokine polypeptides. By “fragment”is intended a protein consisting of only a part of the intact cytokinepolypeptide sequence. The fragment can be a C-terminal deletion orN-terminal deletion of the cytokine polypeptide. By “analogue” isintended an analogue of either the full length polypeptide havingbiological activity or a fragment thereof, that includes a nativesequence and structure having one or more amino acid substitutions,insertions, or deletions. Peptides having one or more peptoids (peptidemimics) are also encompassed by the term analogue (see i.e.,International Publication No. WO 91/04282). By “derivative” is intendedany suitable modification of the native polypeptide or fragmentsthereof, or their respective analogues, such as glycosylation,phosphorylation, or other addition of foreign moieties, so long as theactivity is retained.

[0042] Preferably, naturally or non-naturally occurring variants of acytokine have amino acid sequences that are at least 70%, preferably80%, more preferably, 85%, 90%, 91%, 92%, 93%, 94% or 95% identical tothe amino acid sequence to the reference molecule, for example, thenative human interferon, or to a shorter portion of the referenceinterferon molecule. More preferably, the molecules are 96%, 97%, 98% or99% identical. Percent sequence identity is determined using theSmith-Waterman homology search algorithm using an affine gap search witha gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrixof 62. The Smith-Waterman homology search algorithm is taught in Smithand Waterman, Adv. Appl. Math. (1981) 2:482-489. A variant may, forexample, differ by as few as 1 to 10 amino acid residues, such as 6-10,as few as 5, as few as 4, 3, 2, or even 1 amino aid residue.

[0043] With respect to optimal alignment of two amino acid sequences,the contiguous segment of the variant amino acid sequence may haveadditional amino acid residues or deleted amino acid residues withrespect to the reference amino acid sequence. The contiguous segmentused for comparison to the reference amino acid sequence will include atleast 20 contiguous amino acid residues, and may be 30, 40, 50, or moreamino acid residues. Corrections for sequence identity associated withconservative residue substitutions or gaps can be made (seeSmith-Waterman homology search algorithm).

[0044] The art provides substantial guidance regarding the preparationand use of such variants, as discussed further below. A fragment of acytokine polypeptide will generally include at least about 10 contiguousamino acid residues of the full-length molecule, preferably about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably about 20-50 or more contiguous amino acid residues offull-length cytokine polypeptide.

[0045] For example, conservative amino acid substitutions may be made atone or more predicted, preferably nonessential amino acid residues. A“nonessential” amino acid residue is a residue that can be altered fromthe wild-type sequence of a cytokine, such as an interferon (i.e.,IFN-α, IFN-β, or IFN-γ) without altering its biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Such substitutions would not be made for conserved aminoacid residues, or for amino acid residues residing within a conservedmotif.

[0046] Alternatively, variant cytokine nucleotide sequences can be madeby introducing mutations randomly along all or part of a cytokine codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for cytokine biological activity to identify mutantsthat retain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques described herein.

[0047] Alternatively, the cytokine can be synthesized chemically, by anyof several techniques that are known to those skilled in the peptideart. See, for example, Li et al. (1983) Proc. Natl. Acad. Sci. USA80:2216-2220, Steward and Young (1984) Solid Phase Peptide Synthesis(Pierce Chemical Company, Rockford, Ill.), and Baraney and Merrifield(1980) The Peptides: Analysis, Synthesis, Biology, ed. Gross andMeinhofer, Vol. 2 (Academic Press, New York, 1980), pp. 3-254,discussing solid-phase peptide synthesis techniques; and Bodansky (1984)Principles of Peptide Synthesis (Springer-Verlag, Berlin) and Gross andMeinhofer, eds. (1980) The Peptides: Analysis, Synthesis, Biology, Vol.1 (Academic Press, New York), discussing classical solution synthesis.The cytokine can also be chemically prepared by the method ofsimultaneous multiple peptide synthesis. See, for example, Houghten(1984) Proc. Natl. Acad. Sci. USA 82:5131-5135; and U.S. Pat. No.4,631,211.

[0048] The cytokine used in the methods of the invention can be from anyanimal species including, but not limited to, avian, canine, bovine,porcine, equine, and human. Preferably, the cytokine is from a mammalianspecies when the cytokine is to be used in treatment of a mammalianviral, immunomodulatory, or neurologic disorder of the CNS, brain orspinal cord, and more preferably is from a mammal of the same species asthe mammal undergoing treatment for such a disorder.

[0049] Interferon-β

[0050] The term “IFN-β” as used herein refers to IFN-β or variantsthereof, sometimes referred to as IFN-β-like polypeptides. Human IFN-βvariants, which may be naturally occurring (e.g., allelic variants thatoccur at the IFN-β locus) or recombinantly produced, have amino acidsequences that are the same as, similar to, or substantially similar tothe mature native IFN-β sequence. DNA sequences encoding human IFN-β arealso available in the art. See, for example, Goeddel et al. (1980)Nucleic Acid Res. 8:4057 and Tanigachi et al. (1979) Proc. Japan Acad.Sci. 855:464. Fragments of IFN-β or truncated forms of IFN-β that retaintheir activity are also encompassed. These biologically active fragmentsor truncated forms of IFN-β are generated by removing amino acidresidues from the full-length IFN-β amino acid sequence usingrecombinant DNA techniques well known in the art. IFN-β polypeptides maybe glycosylated or unglycosylated, as it has been reported in theliterature that both the glycosylated and unglycosylated forms of IFN-βshow qualitatively similar specific activities and that, therefore, theglycosyl moieties are not involved in and do not contribute to thebiological activity of IFN-β.

[0051] The IFN-β variants encompassed herein include muteins of thenative mature IFN-β sequence, wherein one or more cysteine residues thatare not essential to biological activity have been deliberately deletedor replaced with other amino acids to eliminate sites for eitherintermolecular crosslinking or incorrect intramolecular disulfide bondformation. IFN-β variants of this type include those containing aglycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine,histidine, tryptophan, serine, threonine, or methionine substituted forthe cysteine found at amino acid 17 of the mature native amino acidsequence. Serine and threonine are the more preferred replacementsbecause of their chemical analogy to cysteine. Serine substitutions aremost preferred. For example, an IFN-β variant can comprise a serineresidue replacing the cysteine found at amino acid 17 of the maturenative sequence. Cysteine 17 may also be deleted using methods known inthe art (see, for example, U.S. Pat. No. 4,588,585, herein incorporatedby reference), resulting in a mature IFN-β mutein that is one amino acidshorter than the native mature IFN-β. Thus, IFN-β variants with one ormore mutations that improve, for example, their pharmaceutical utilityare also encompassed by the present invention.

[0052] The skilled artisan will appreciate that additional changes canbe introduced by mutation into the nucleotide sequences encoding IFN-β,thereby leading to changes in the IFN-β amino acid sequence, withoutaltering the biological activity of the interferon. Thus, an isolatednucleic acid molecule encoding an IFN-β variant having a sequence thatdiffers from human IFN-β can be created by introducing one or morenucleotide substitutions, additions, or deletions into the correspondingnucleotide sequence disclosed herein, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedIFN-β. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Such IFN-βvariants are also encompassed by the present invention. Variants ofIFN-β are described in European Patent Application No. 18545981, andU.S. Pat. Nos. 4,518,584, 4,588,585, and 4,737,462, all of which areincorporated herein by reference.

[0053] Biologically active IFN-β variants encompassed by the inventionalso include IFN-β polypeptides that have covalently linked with, forexample, polyethylene glycol (PEG) or albumin.

[0054] Biologically active variants of IFN-β encompassed by theinvention should retain IFN-β activities, particularly the ability tobind to IFN-p receptors or retain immunomodulatory or anti-viralactivities. In some embodiments, the IFN-β variant retains at leastabout 25%, about 50%, about 75%, about 85%, about 90%, about 95%, about98%, about 99% or more of the biological activity of the native IFN-βpolypeptide. IFN-β variants whose activity is increased in comparisonwith the activity of the native IFN-β polypeptide are also encompassed.The biological activity of IFN-p variants can be measured by any methodknown in the art. Examples of such assays can be found in Fellous et aL(1982) Proc. Natl. Acad. Sci USA 79:3082-3086; Czemiecki et al. (1984)J. Virol. 49(2):490-496; Mark et al (1984) Proc. Natl Acad. Sci. USA81:5662-5666; Branca et al. (1981) Nature 277:221-223; Williams et al.(1979) Nature 282:582-586; Herberman et aL (1979) Nature 277:221-223;and Anderson etal. (1982) J. Biol. Chem. 257(19):11301-11304.

[0055] Non-limiting examples of IFN-β polypeptides and IFN-β variantpolypeptides encompassed by the invention are set forth in Nagata et al.(1980) Nature 284:316-320; Goeddel et al. (1980) Nature 287:411-416 ;Yelverton etaL (1981) Nucleic Acids Res. 9:731-741; Streuli et al.(1981) Proc. Natl. Acad. Sci. U.S.A. 78:2848-2852; EP028033B1, andEP109748B1. See also U.S. Pat. Nos. 4,518,584; 4,569,908; 4,588,585;4,738,844; 4,753,795; 4,769,233; 4,793,995; 4,914,033; 4,959,314;5,545,723; and 5,814,485. These disclosures are herein incorporated byreference. These citations also provide guidance regarding residues andregions of the IFN-β polypeptide that can be altered without the loss ofbiological activity.

[0056] In one embodiment of the present invention, the IFN-β used in themethods of the invention is the mature native human IFN-β polypeptide.In another embodiment, the IFN-β is the mature IFN-β C17S polypeptide.However, the present invention encompasses other embodiments where theIFN-β is any biologically active IFN-β polypeptide or variant asdescribed elsewhere herein.

[0057] In some embodiments of the present invention, the IFN-β isrecombinantly produced. By “recombinantly produced IFN-β” is intendedIFN-β that has comparable biological activity to native IFN-β and thathas been prepared by recombinant DNA techniques. IFN-β can be producedby culturing a host cell transformed with an expression vectorcomprising a nucleotide sequence that encodes an IFN-β polypeptide. Thehost cell is one that can transcribe the nucleotide sequence and producethe desired protein, and can be prokaryotic (for example, E. coli) oreukaryotic (for example a yeast, insect, or mammalian cell). Examples ofrecombinant production of IFN-β are given in Mantei et al. (1982) Nature297:128; Ohno et al. (1982) Nucleic Acids Res. 10:967; Smith et al.(1983) Mol. Cell. Biol. 3:2156, and U.S. Pat. No. 4,462,940, 5,702,699,and 5,814,485; herein incorporated by reference.

[0058] Interferon-α

[0059] The term “IFN-α” as used herein refers to IFN-α or variantsthereof, sometimes referred to as IFN-α-like polypeptides. Human alphainterferons comprise a family of about 30 protein species, encoded by atleast 14 different genes and about 16 alleles. Such IFN-α polypeptidesinclude IFN-αa, IFN-αB, IFN-αC, IFN-αD, IFN-αH, IFN-αJ, IFN-αJ1, IFN-αJ2and IFN-αK. Human IFN-α variants, which may be naturally occurring(e.g., allelic variants that occur at the IFN-α locus) or recombinantlyproduced, have amino acid sequences that are the same as, similar to, orsubstantially similar to the mature native IFN-α sequence. DNA sequencesencoding human IFN-α are also available in the art. See, for example,Goeddel et al. (1981) Nature 290:20-26 (Genbank Accession No. V00551J00209); Nagata et al. (1980) Nature 284:3126-320; Bowden et al. (1984)Gene 27:87-99 (Genbank Accession No. NM_(—)000605); and Ohara et al.(1987) FEBS Letters 211:78-82; all of which are herein incorporated byreference. Fragments of IFN-α or truncated forms of IFN-α that retaintheir activity are also encompassed. These biologically active fragmentsor truncated forms of IFN-α are generated by removing amino acidresidues from the full-length IFN-α amino acid sequence usingrecombinant DNA techniques well known in the art. IFN-α polypeptides mayfurther be glycosylated or unglycosylated.

[0060] The skilled artisan will appreciate that additional changes canbe introduced by mutation into the nucleotide sequences encoding IFN-α,thereby leading to changes in the IFN-α amino acid sequence, withoutaltering the biological activity of the interferon. Thus, an isolatednucleic acid molecule encoding an IFN-α variant having a sequence thatdiffers from human IFN-α can be created by introducing one or morenucleotide substitutions, additions, or deletions into the correspondingnucleotide sequence disclosed herein, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedIFN-α. Mutations can be introduced by standard techniques. Such variantsof IFN-α, include, for example, IFN-α-2a (Roferon-A™), IFN-α-2b (IntronA™), and IFN-αcon-1 (Infergen™). Another variant useful in the methodsof the present invention is IFN-α2a, which is disclosed in, for example,EP 43980; Meada et al. (1980) PNAS 77:7010; and Levy et al. (1981) PNAS78:6186; all of which are herein incorporated by reference. Further,variants of IFN-α can be found, for example, in U.S. Pat. No. 5,676,942,herein incorporated by reference. These citations also provide guidanceregarding residues and regions of the IFN-α polypeptide that can bealtered without the loss of biological activity.

[0061] Biologically active IFN-α variants encompassed by the inventionalso include IFN-α polypeptides that have covalently linked with, forexample, polyethylene glycol (PEG) or albumin. See, for example, U.S.Pat. No. 5,762,923, herein incorporated by reference.

[0062] Biologically active variants of IFN-α encompassed by theinvention should retain IFN-α activities, particularly the ability tobind to IFN-α receptors or retain immunomodulatory, antiviral, oranit-proliferative activities. In some embodiments, the IFN-α variantretains at least about 25%, about 50%, about 75%, about 85%, about 90%,about 95%, about 98%, about 99% or more of the biological activity ofthe native IFN-α polypeptide. IFN-α variants whose activity is increasedin comparison with the activity of the native IFN-α polypeptide are alsoencompassed. The biological activity of IFN-α variants can be measuredby any method known in the art. Examples of such assays are describeabove.

[0063] In one embodiment of the present invention, the IFN-α used in themethods of the invention is the mature native human IFN-α polypeptide.However, the present invention encompasses other embodiments where theIFN-α is any biologically active IFN-α polypeptide or variant asdescribed elsewhere herein.

[0064] In some embodiments of the present invention, the IFN-α isrecombinantly produced. By “recombinantly produced IFN-α” is intendedIFN-α that has comparable biological activity to native IFN-α and thathas been prepared by recombinant DNA techniques. IFN-α can be producedby culturing a host cell transformed with an expression vectorcomprising a nucleotide sequence that encodes an IFN-α polypeptide. Thehost cell is one that can transcribe the nucleotide sequence and producethe desired protein, and can be prokaryotic (for example, E. coli) oreukaryotic (for example a yeast, insect, or mammalian cell). Details ofthe cloning of interferon-cDNA and the direct expression thereof,especially in E. coli, have in the meantime been the subject of manypublications. Thus, for example, the preparation of recombinantinterferons is known. See, for example, (1982) Nature 295: 503-508;(1980) Nature 284: 316-320; (1981) Nature 290: 20-26; (1980) NucleicAcids Res. 8: 4057-4074, as well as from European Patents Nos. 32134,43980 and 211 148. Further examples of recombinant production of IFN-α-2are provided in Nagata et al. (1980) Nature 284:316 and European Patent32,134. All of these references are herein incorporated by reference.

[0065] Interferon-γ

[0066] The term “IFN-γ” as used herein refers to IFN-γ or variantsthereof, sometimes referred to as IFN-γ-like polypeptides. IFN-γ is aglycoprotein whose mature form has 143 amino acids and a molecularweight of about 63-73 kilodaltons. The amino acid sequence of IFN-γ canbe found in, for example, U.S. Pat. No. 6,046,034, herein incorporatedby reference. Human IFN-γ variants, which may be naturally occurring(e.g., allelic variants that occur at the IFN-γ locus) or recombinantlyproduced, have amino acid sequences that are the same as, similar to, orsubstantially similar to the mature native IFN-γ sequence. DNA sequencesencoding human IFN-γ are also available in the art. See, for example,Grey et al. (1983) Proc. Natl. Acad. Sci. USA 80:5842-5846, hereinincorporated by reference. Fragments of IFN-γ or truncated forms ofIFN-γ that retain their activity are also encompassed. Thesebiologically active fragments or truncated forms of IFN-γ are generatedby removing amino acid residues from the full-length IFN-γ amino acidsequence using recombinant DNA techniques well known in the art. IFN-γpolypeptides may be glycosylated or unglycosylated.

[0067] The IFN-γ variants encompassed herein include muteins of thenative mature IFN-γ sequence. Thus, IFN-γ variants with one or moremutations that improve, for example, their pharmaceutical utility arealso encompassed by the present invention.

[0068] Such IFN-γ variants are also encompassed by the presentinvention. Variants of IFN-γ are well known in the art. For example,U.S. Pat. No. 5,770,191, herein incorporated by reference, disclosespeptides comprising the C-terminus of IFN-γ that retain the biologicalactivity of the mature IFN-γ. Additionally, in EP 0 306870 A2, variantsof human IFN-γ were identified whose activity was significantlyincreased by deleting the C-terminal 7-11 amino acids. In addition, WO92-08737 discloses a variant of recombinant human IFN-γ(IFN-γ C-10L)that has increased biological activity. Further variants of IFN-γ can befound in, for example, U.S. Pat. No. 5,690,925 and U.S. Pat. No.6,046,034 both of which provide guidance as to the amino acidsubstitutions and deletions that can be made in IFN-γ without losingbiological activity. Each of these references is herein incorporated byreference. The above examples represent non-limiting examples of IFN-γpolypeptides and IFN-γ variant polypeptides encompassed by theinvention. These citations also provide guidance regarding residues andregions of the IFN-γ polypeptide that can be altered without the loss ofbiological activity.

[0069] Biologically active IFN-γ variants encompassed by the inventionalso include IFN-γ polypeptides that have covalently linked with, forexample, polyethylene glycol (PEG) or albumin.

[0070] Biologically active variants of IFN-γ encompassed by theinvention should retain IFN-γ activities, particularly the ability tobind to IFN-γ receptors or retain immunomodulatory, antiviral, orantiproliferative activities. In some embodiments, the IFN-γ variantretains at least about 25%, about 50%, about 75%, about 85%, about 90%,about 95%, about 98%, about 99% or more of the biological activity ofthe native IFN-γ polypeptide. IFN-γ variants whose activity is increasedin comparison with the activity of the native IFN-γ polypeptide are alsoencompassed. The biological activity of IFN-γ variants can be measuredby any method known in the art. Examples of such assays are describedabove.

[0071] In one embodiment of the present invention, the IFN-γ used in themethods of the invention is the mature native human IFN-γ polypeptide.However, the present invention encompasses other embodiments where theIFN-γ is any biologically active IFN-γ polypeptide or variant asdescribed elsewhere herein.

[0072] In some embodiments of the present invention, the IFN-γ isrecombinantly produced. By “recombinantly produced IFN-γ” is intendedIFN-γ that has comparable biological activity to native IFN-γ and thathas been prepared by recombinant DNA techniques. IFN-γ can be producedby culturing a host cell transformed with an expression vectorcomprising a nucleotide sequence that encodes an IFN-γ polypeptide. Thehost cell is one that can transcribe the nucleotide sequence and producethe desired protein, and can be prokaryotic (for example, E. coli) oreukaryotic (for example a yeast, insect, or mammalian cell). Examples ofrecombinant production of IFN-γ are given in U.S Pat. Nos. 6,046,034 and5,690,925; both of which are herein incorporated by reference.

[0073] Pharmaceutical Composition

[0074] Increases in the amount of cytokine in the CNS, brain, and/orspinal cord to a therapeutically effective level may be obtained viaadministration of a pharmaceutical composition including atherapeutically effective dose of this cytokine. By “therapeuticallyeffective dose” is intended a dose of cytokine that achieves the desiredgoal of increasing the amount of this cytokine in a relevant portion ofthe CNS, brain, and/or spinal cord to a therapeutically effective levelenabling a desired biological activity of the cytokine.

[0075] The invention is, in particular, directed to a composition thatcan be employed for delivery of a cytokine to the CNS, brain, and/orspinal cord upon administration to tissue innervated by the olfactoryand/or trigeminal nerves. The composition can include, for example, anypharmaceutically acceptable additive, carrier, or adjuvant that issuitable for administering a cytokine to tissue innervated by theolfactory and/or trigeminal nerves. Preferably, the pharmaceuticalcomposition can be employed in diagnosis, prevention, or treatment of adisease, disorder, or injury of the CNS, brain, and/or spinal cord.Preferably, the composition includes a cytokine in combination with apharmaceutical carrier, additive, and/or adjuvant that can promote thetransfer of the cytokine within or through tissue innervated by theolfactory and/or trigeminal nerves. Alternatively, the cytokine may becombined with substances that may assist in transporting the cytokine tosites of nerve cell damage. The composition can include one or severalcytokines.

[0076] The composition typically contains a pharmaceutically acceptablecarrier mixed with the cytokine and other components in thepharmaceutical composition. By “pharmaceutically acceptable carrier” isintended a carrier that is conventionally used in the art to facilitatethe storage, administration, and/or the healing effect of the cytokine.A carrier may also reduce any undesirable side effects of the cytokine.A suitable carrier should be stable, i.e., incapable of reacting withother ingredients in the formulation. It should not produce significantlocal or systemic adverse effect in recipients at the dosages andconcentrations employed for treatment. Such carriers are generally knownin the art.

[0077] Suitable carriers for this invention include those conventionallyused for large stable macromolecules such as albumin, gelatin, collagen,polysaccharide, monosaccharides, polyvinylpyrrolidone, polylactic acid,polyglycolic acid, polymeric amino acids, fixed oils, ethyl oleate,liposomes, glucose, sucrose, lactose, mannose, dextrose, dextran,cellulose, mannitol, sorbitol, polyethylene glycol (PEG), and the like.

[0078] Water, saline, aqueous dextrose, and glycols are preferred liquidcarriers, particularly (when isotonic) for solutions. The carrier can beselected from various oils, including those of petroleum, animal,vegetable or synthetic origin, for example, peanut oil, soybean oil,mineral oil, sesame oil, and the like. Suitable pharmaceuticalexcipients include starch, cellulose, talc, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate,sodium stearate, glycerol monostearate, sodium chloride, dried skimmilk, glycerol, propylene glycol, water, ethanol, and the like. Thecompositions can be subjected to conventional pharmaceutical expedients,such as sterilization, and can contain conventional pharmaceuticaladditives, such as preservatives, stabilizing cytokines, wetting, oremulsifying agents, salts for adjusting osmotic pressure, buffers, andthe like.

[0079] A composition formulated for intranasal delivery may optionallycomprise an odorant. An odorant agent is combined with the cytokine toprovide an odorliferous sensation, and/or to encourage inhalation of theintranasal preparation to enhance delivery of the active cytokine to theolfactory neuroepithelium. The odorliferous sensation provided by theodorant agent may be pleasant, obnoxious, or otherwise malodorous. Theodorant receptor neurons are localized to the olfactory epithelium that,in humans, occupies only a few square centimeters in the upper part ofthe nasal cavity. The cilia of the olfactory neuronal dendrites whichcontain the receptors are fairly long (about 30-200 um). A 10-30 μmlayer of mucus envelops the cilia that the odorant agent must penetrateto reach the receptors. See Snyder et al. (1998) J Biol. Chem.263:13972-13974. Use of a lipophillic odorant agent having moderate tohigh affinity for odorant binding protein (OBP) is preferred. OBP has anaffinity for small lipophillic molecules found in nasal secretions andmay act as a carrier to enhance the transport of a lipophillic odorantsubstance and cytokines to the olfactory receptor neurons. It is alsopreferred that an odorant agent is capable of associating withlipophillic additives such as liposomes and micelles within thepreparation to farther enhance delivery of the cytokines by means of OBPto the olfactory neuroepithelium. OBP may also bind directly tolipophillic agents to enhance transport of the cytokines to olfactoryneural receptors.

[0080] Suitable odorants having a high affinity for OBP includeterpanoids such as cetralva and citronellol, aldehydes such as amylclnnamaldehyde and hexyl cinnamaldehyde, esters such as octylisovalerate, jasmines such as C1S-jasmine and jasmal, and musk 89. Othersuitable odorant agents include those which may be capable ofstimulating odorant-sensitive enzymes such as aderrylate cyclase andguanylate cyclase, or which may be capable of modifying ion channelswithin the olfactory system to enhance absorption of the cytokine.

[0081] Other acceptable components in the composition include, but arenot limited to, pharmaceutically acceptable agents that modifyisotonicity, including water, salts, sugars, polyols, amino acids andbuffers, such as, phosphate, citrate, succinate, acetate, and otherorganic acids or their salts. Typically, the pharmaceutically acceptablecarrier also includes one or more stabilizers, reducing agents,anti-oxidants and/or anti-oxidant chelating agents. The use of buffers,stabilizers, reducing agents, anti-oxidants and chelating agents in thepreparation of protein based compositions, particularly pharmaceuticalcompositions, is well known in the art. See Wang et al. (1980) J.Parent. Drug Assn., 34(6):452-462; Wang et al. (1988) J Parent. Sci. andTech. 42:S4-S26 (Supplement); Lachman, et al. (1968) Drug and CosmeticIndustry, 102(1): 36-38, 40 and 146-148; Akers, M. J. (1988) J Parent.Sci. and Tech., 36(5):222-228; and Colowick et al. Methods inEnzymology, Vol. XXV, p. 185-188.

[0082] Suitable buffers include acetate, adipate, benzoate, citrate,lactate, maleate, phosphate, tartarate, borate, tri(hydroxymethylaminomethane), succinate, glycine, histidine, the salts of various aminoacids, or the like, or combinations thereof. See Wang (1980) supra atpage 455. Suitable salts and isotonicifiers include sodium chloride,dextrose, mannitol, sucrose, trehalose, or the like. Where the carrieris a liquid, it is preferred that the carrier is hypotonic or isotonicwith oral, conjunctival or dermal fluids and have a pH within the rangeof 4.5-8.5. Where the carrier is in powdered form, it is preferred thatthe carrier is also within an acceptable non-toxic pH range.

[0083] Suitable reducing agents, which maintain the reduction of reducedcysteines, include dithiothreitol (DTT also known as Cleland's reagent)or dithioerythritol at 0.01% to 0.1% wt/wt; acetylcysteine or cysteineat 0.1% to 0.5% (pH 2-3); and thioglycerol at 0.1% to 0.5% (pH 3.5 to7.0) and glutathione. See Akers (1988) supra at pages 225 to 226.Suitable antioxidants include sodium bisulfite, sodium sulfite, sodiummetabisulfite, sodium thiosulfate, sodium formaldehyde sulfoxylate, andascorbic acid. See Akers (1988) supra at pages 225. Suitable chelatingagents, which chelate trace metals to prevent the trace metal catalyzedoxidation of reduced cysteines, include citrate, tartarate,ethylenediaminetetraacetic acid (EDTA) in its disodium, tetrasodium, andcalcium disodium salts, and diethylenetriamine pentaacetic acid (DTPA).See, e.g., Wang (1980) supra at pages 457-458 and 460-461, and Akers(1988) supra at pages 224-227.

[0084] The composition can include one or more preservatives such asphenol, cresol, p-aminobenzoic acid, BDSA, sorbitrate, chlorhexidine,benzalkonium chloride, or the like. Suitable stabilizers includecarbohydrates such as trehalose or glycerol. The composition can includea stabilizer such as one or more of microcrystalline cellulose,magnesium stearate, mannitol, sucrose to stabilize, for example, thephysical form of the composition; and one or more of glycine, arginine,hydrolyzed collagen, or protease inhibitors to stabilize, for example,the chemical structure of the composition. Suitable suspending additivesinclude carboxymethyl cellulose, hydroxypropyl methylcellulose,hyaluronic acid, alginate, chondroitin sulfate, dextran, maltodextrin,dextran sulfate, or the like. The composition can include an emulsifiersuch as polysorbate 20, polysorbate 80, pluronic, triolein, soybean oil,lecithins, squalene and squalanes, sorbitan treioleate, or the like. Thecomposition can include an antimicrobial such as phenylethyl alcohol,phenol, cresol, benzalkonium chloride, phenoxyethanol, chlorhexidine,thimerosol, or the like. Suitable thickeners include naturalpolysaccharides such as mannans, arabinans, alginate, hyaluronic acid,dextrose, or the like; and synthetic ones like the PEG hydrogels of lowmolecular weight and aforementioned suspending cytokines.

[0085] The composition can include an adjuvant such as cetyl trimethylammonium bromide, BDSA, cholate, deoxycholate, polysorbate 20 and 80,fusidic acid, or the like, and in the case of DNA delivery, preferably,a cationic lipid. Suitable sugars include glycerol, threose, glucose,galactose, mannitol, and sorbitol. A suitable protein is human serumalbumin.

[0086] Preferred compositions include one or more of a solubilityenhancing additive, preferably a cyclodextrin; a hydrophilic additive,preferably a monosaccharride or oligosaccharide; an absorption promotingadditive, preferably a cholate, a deoxycholate, a fusidic acid, or achitosan; a cationic surfactant, preferably a cetyl trimethyl ammoniumbromide; a viscosity enhancing additive, preferably to promote residencetime of the composition at the site of administration, preferably acarboxymethyl cellulose, a maltodextrin, an alginic acid, a hyaluronicacid, or a chondroitin sulfate; or a sustained release matrix,preferably a polyanhydride, a polyorthoester, a hydrogel, a particulateslow release depo system, preferably a polylactide co-glycolides (PLG),a depo foam, a starch microsphere, or a cellulose derived buccal system;a lipid-based carrier, preferably an emulsion, a liposome, a niosomes,or a micelles. The composition can include a bilayer destabilizingadditive, preferably a phosphatidyl ethanolamine; a fusogenic additive,preferably a cholesterol hemisuccinate.

[0087] Other preferred compositions for sublingual administrationincluding, for example, a bioadhesive to retain the cytokinesublingually; a spray, paint, or swab applied to the tongue; retaining aslow dissolving pill or lozenge under the tongue; or the like. Otherpreferred compositions for transdermal administration include abioadhesive to retain the cytokine on or in the skin; a spray, paint,cosmetic, or swab applied to the skin; or the like.

[0088] These lists of carriers and additives is by no means complete anda worker skilled in the art can choose excipients from the GRAS(generally regarded as safe) list of chemicals allowed in thepharmaceutical preparations and those that are currently allowed intopical and parenteral formulations.

[0089] For the purposes of this invention, the pharmaceuticalcomposition comprising the cytokine can be formulated in a unit dosageand in a form such as a solution, suspension, or emulsion. The cytokinemay be administered to tissue innervated by the trigeminal and/orolfactory neurons as a powder, a granule, a solution, a cream, a spray(e.g., an aerosol), a gel, an ointment, an infusion, an injection, adrop, or sustained-release composition, such as a polymer disk. Forbuccal administration, the compositions can take the form of tablets orlozenges formulated in a conventional manner. For administration to theeye or other external tissues, e.g., mouth and skin, the compositionscan be applied to the infected part of the body of the patient as atopical ointment or cream. The compounds can be presented in anointment, for instance with a water-soluble ointment base, or in acream, for instance with an-oil-in water cream base. For conjunctivalapplications, the cytokine can be administered in biodegradable ornon-degradable ocular inserts. The drug may be released by matrixerosion or passively through a pore as in ethylene-vinylacetate polymerinserts. For other mucosal administrations, such as sublingual, powderdiscs may be placed under the tongue and active delivery systems may forin situ by slow hydration as in the formulation of liposomes from driedlipid mixtures or pro-liposomes.

[0090] Other preferred forms of compositions for administration includea suspension of a particulate, such as an emulsion, a liposome, aninsert that releases the cytokine slowly, and the like. The powder orgranular forms of the pharmaceutical composition may be combined with asolution and with a diluting, dispersing, or surface-active cytokine.Additional preferred compositions for administration include abioadhesive to retain the cytokine at the site of administration; aspray, paint, or swab applied to the mucosa or epithelium; a slowdissolving pill or lozenge; or the like. The composition can also be inthe form of lyophilized powder, which can be converted into a solution,suspension, or emulsion before administration. The pharmaceuticalcomposition including cytokine is preferably sterilized by membranefiltration and is stored in unit-dose or multi-dose containers such assealed vials or ampoules.

[0091] The method for formulating a pharmaceutical composition isgenerally known in the art. A thorough discussion of formulation andselection of pharmaceutically acceptable carriers, stabilizers, andisomolytes can be found in Remington's Pharmaceutical Sciences (18^(th)ed.; Mack Publishing Company, Eaton, Pa., 1990), herein incorporated byreference.

[0092] The cytokine of the present invention can also be formulated in asustained-release form to prolong the presence of the pharmaceuticallyactive cytokine in the treated mammal, generally for longer than oneday. Many methods of preparation of a sustained-release formulation areknown in the art and are disclosed in Remington's PharmaceuticalSciences (18^(th) ed.; Mack Publishing Company, Eaton, Pennsylvania,1990), herein incorporated by reference.

[0093] Generally, the cytokine can be entrapped in semipermeablematrices of solid hydrophobic polymers. The matrices can be shaped intofilms or microcapsules. Examples of such matrices include, but are notlimited to, polyesters, copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al. (1983) Biopolymers 22:547-556),polylactides (U.S. Pat. No. ,773,919 and EP 58,481), polylactatepolyglycolate (PLGA) such as polylactide-co-glycolide (see, for example,U.S. Pat. Nos. 4,767,628 and 5,654,008), hydrogels (see, for example,Langer et al. (1981) J Biomed. Mater. Res. 15:167-277; Langer (1982)Chem. Tech. 12:98-105), non-degradable ethylene-vinyl acetate (e.g.ethylene vinyl acetate disks and poly(ethylene-co-vinyl acetate)),degradable lactic acid-glycolic acid copolyers such as the LupronDepot™, poly-D-(-)-3-hydroxybutyric acid (EP 133,988), hyaluronic acidgels (see, for example, U.S. Pat. No. 4,636,524), alginic acidsuspensions, and the like.

[0094] Suitable microcapsules can also include hydroxymethylcellulose orgelatin-microcapsules and polymethyl methacrylate microcapsules preparedby coacervation techniques or by interfacial polymerization. See the PCTpublication WO 99/24061 entitled “Method for Producing Sustained-releaseFormulations,” wherein a protein is encapsulated in PLGA microspheres,herein incorporated by reference. In addition, microemulsions orcolloidal drug delivery systems such as liposomes and albuminmicrospheres, may also be used. See Remington's Pharmaceutical Sciences(18^(th) ed.; Mack Publishing Company Co., Eaton, Pa., 1990). Otherpreferred sustained-release compositions employ a bioadhesive to retainthe cytokine at the site of administration.

[0095] Among the optional substances that may be combined with thecytokine in the pharmaceutical composition are lipophilic substancesthat can enhance absorption of the cytokine through the mucosa orepithelium of the nasal cavity, or along a neural, lymphatic, orperivascular pathway to damaged nerve cells in the CNS. The cytokine maybe mixed with a lipophilic adjuvant alone or in combination with acarrier, or may be combined with one or several types of micelle orliposome substances. Among the preferred lipophilic substances arecationic liposomes included of one or more of the following:phosphatidyl choline, lipofectin, DOTAP, a lipid-peptoid conjugate, asynthetic phospholipid such as phosphatidyl lysine, or the like. Theseliposomes may include other lipophilic substances such as gangliosidesand phosphatidylserine (PS). Also preferred are micellar additives suchas GM-1 gangliosides and phosphatidylserine (PS), which may be combinedwith the cytokine either alone or in combination. GM-1 ganglioside canbe included at 1-10 mole percent in any liposomal compositions or inhigher amounts in micellar structures. Protein cytokines can be eitherencapsulated in particulate structures or incorporated as part of thehydrophobic portion of the structure depending on the hydrophobicity ofthe active cytokine.

[0096] One preferred liposomal formulation employs Depofoam. A cytokinecan be encapsulated in multivesicular liposomes, as disclosed in the WOpublication 99/12522 entitled “High and Low Load Formulations of IGF-Iin Multivesicular Liposomes,” herein incorporated by reference. The meanresidence time of cytokine at the site of administration can beprolonged with a Depofoam composition.

[0097] Administering the Cytokine

[0098] According to this embodiment of the invention, the total amountof cytokine administered per dose should be in a range sufficient todelivery a biologically relevant amount of the cytokine (i.e., an amountsufficient to produce a therapeutical effect). The pharmaceuticalcomposition having a unit dose of cytokine can be in the form ofsolution, suspension, emulsion, or a sustained-release formulation. Thetotal volume of one dose of the pharmaceutical composition can rangefrom about 10 μl to about 100 μl, for example, for nasal administration.It is apparent that the suitable volume can vary with factors such asthe size of the tissue to which the cytokine is administered and thesolubility of the components in the composition.

[0099] It is recognized that the total amount of cytokine administeredas a unit dose to a particular tissue will depend upon the type ofpharmaceutical composition being administered, that is whether thecomposition is in the form of, for example, a solution, a suspension, anemulsion, or a sustained-release formulation. For example, where thepharmaceutical composition comprising a therapeutically effective amountof cytokine is a sustained-release formulation, cytokine is administeredat a higher concentration. Needle-free subcutaneous administration to anextranasal tissue innervated by the trigeminal nerve may be accomplishedby use of a device which employs a supersonic gas jet as a power sourceto accelerate an agent that is formulated as a powder or a microparticleinto the skin. The characteristics of such a delivery method will bedetermined by the properties of the particle, the formulation of theagent and the gas dynamics of the delivery device. Similarly, thesubcutaneous delivery of an aqueous composition can be accomplished in aneedle-free manner by employing a gas-spring powered hand held device toproduce a high force jet of fluid capable of penetrating the skin.Alternatively, a skin-patch formulated to mediate a sustained release ofa composition can be employed for the transdermal delivery of aneuroregulatory agent to a tissue innervated by the trigeminal nerve.Where the pharmaceutical composition comprises a therapeuticallyeffective amount of an agent, or a combination of agents, in asustained-release formulation, the agent(s) is/are administered at ahigher concentration.

[0100] It should be apparent to a person skilled in the art thatvariations may be acceptable with respect to the therapeuticallyeffective dose and frequency of the administration a cytokine in thisembodiment of the invention. The amount of the cytokine administeredwill be inversely correlated with the frequency of administration.Hence, an increase in the concentration of cytokine in a singleadministered dose, or an increase in the mean residence time in the caseof a sustained-release form of cytokine, generally will be coupled witha decrease in the frequency of administration.

[0101] In the practice of the present invention, additional factorsshould be taken into consideration when determining the therapeuticallyeffective dose of cytokine and frequency of its administration. Suchfactors include, for example, the size of the tissue, the area of thesurface of the tissue, the severity of the disease or disorder, and theage, height, weight, health, and physical condition of the individual tobe treated. Generally, a higher dosage is preferred if the tissue islarger or the disease or disorder is more severe.

[0102] Some minor degree of experimentation may be required to determinethe most effective dose and frequency of dose administration, this beingwell within the capability of one skilled in the art once apprised ofthe present disclosure.

[0103] For the treatment of a disorder of the CNS in a human, includingneurologic, viral, proliferative or immunomodulatory disorders, atherapeutically effective amount or dose of a cytokine is about 0.14nmol/kg of brain weight to about 138 nmol/kg brain weight and about 0.14nmol/kg of brain weight to about 69 nmol/kg of brain weight. In someregimens, therapeutically effective doses for administration of acytokine include about 0. 13, 0.2, 0.4, 0.6, 0.8, 1.0, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, or 140 nmoles per kg of brainweight. For the treatment of a disorder of the CNS in a human, includingneurologic, viral, proliferative or immunomodulatory disorders, thetherapeutically effective amount or dose of IFN-β or biologically activevariant thereof is about 0.14 nmol/kg of brain weight to about 138nmol/kg of brain weight and about 0.14 nmol/kg of brain weight to about69 nmol/kg of brain weight. In some regimens, therapeutically effectivedoses for administration of IFN-β include about 0.13, 0.2, 0.4, 0.6,0.8, 1.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 140nmoles per kg of brain weight.

[0104] It is further recognized that the therapeutically effectiveamount or dose of a cytokine to a human may be lower when the cytokineis administered via the nasal lymphatics to various tissues of the headand neck for the treatment or prevention of disorders or diseasescharacterized by immune and inflammatory responses (i.e., diseasesresulting in acute or chronic inflammation and/or infiltration bylymphocytes). In these embodiments, while the cytokine can beadministered in the dosage range provided above, the cytokine may alsobe administered from about 0.02 to about 138 pmol/kg of brain weight.Alternatively, the cytokine may be administered from about 0.02, 0.03,0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, or 140 pmol per kg of brainweight. Similarly, when the cytokine is IFN-β, the dosage range may alsobe from about 0.02 to about 138 pmol/kg of brain weight. Alternatively,the cytokine may be administered from about 0.02, 0.03, 0.08, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, or 140 pmol per kg of brain weight.

[0105] These doses depend on factors including the efficiency with whichcytokine IFN-β is transported to the CNS or lymphatic system. A largertotal dose can be delivered by multiple administrations of the agent.

[0106] Intermittent Dosing

[0107] In another embodiment of the invention, the pharmaceuticalcomposition comprising the therapeutically effective dose of cytokine isadministered intermittently. By “intermittent administration” isintended administration of a therapeutically effective dose of cytokine,followed by a time period of discontinuance, which is then followed byanother administration of a therapeutically effective dose, and soforth. Administration of the therapeutically effective dose may beachieved in a continuous manner, as for example with a sustained-releaseformulation, or it may be achieved according to a desired daily dosageregimen, as for example with one, two, three or more administrations perday. By “time period of discontinuance” is intended a discontinuing ofthe continuous sustained-released or daily administration of cytokine.The time period of discontinuance may be longer or shorter than theperiod of continuous sustained-release or daily administration. Duringthe time period of discontinuance, the cytokine level in the relevanttissue is substantially below the maximum level obtained during thetreatment. The preferred length of the discontinuance period depends onthe concentration of the effective dose and the form of cytokine used.The discontinuance period can be at least 2 days, preferably is at least4 days, more preferably is at least 1 week and generally does not exceeda period of 4 weeks. When a sustained-release formulation is used, thediscontinuance period must be extended to account for the greaterresidence time of cytokine at the site of injury. Alternatively, thefrequency of administration of the effective dose of thesustained-release formulation can be decreased accordingly. Anintermittent schedule of administration of cytokine can continue untilthe desired therapeutic effect, and ultimately treatment of the diseaseor disorder, is achieved.

[0108] In yet another embodiment, intermittent administration of thetherapeutically effective dose of cytokine is cyclic. By “cyclic” isintended intermittent administration accompanied by breaks in theadministration, with cycles ranging from about 1 month to about 2, 3, 4,5, or 6 months. For example, the administration schedule might beintermittent administration of the effective dose of cytokine, wherein asingle short-term dose is given once per week for 4 weeks, followed by abreak in intermittent administration for a period of 3 months, followedby intermittent administration by administration of a single short-termdose given once per week for 4 weeks, followed by a break inintermittent administration for a period of 3 months, and so forth. Asanother example, a single short-term dose may be given once per week for2 weeks, followed by a break in intermittent administration for a periodof 1 month, followed by a single short-term dose given once per week for2 weeks, followed by a break in intermittent administration for a periodof 1 month, and so forth. A cyclic intermittent schedule ofadministration of cytokine to subject may continue until the desiredtherapeutic effect, and ultimately treatment of the disorder or disease,is achieved.

[0109] Neuronal Transport

[0110] One embodiment of the present method includes administration ofthe cytokine to the subject in a manner such that the cytokine istransported to the lymphatic system, the lacrimal gland, CNS, brain,and/or spinal cord along a neural pathway. A neural pathway includestransport within or along a neuron, through or by way of lymphaticsrunning with a neuron, through or by way of a perivascular space of ablood vessel running with a neuron or neural pathway, through or by wayof an adventitia of a blood vessel running with a neuron or neuralpathway, or through an hemangiolymphatic system. The invention preferstransport of a cytokine by way of a neural pathway, rather than throughthe circulatory system, so that cytokines that are unable to, or onlypoorly, cross the blood-brain barrier from the bloodstream into thebrain can be delivered to the lymphatic system, CNS, brain, and/orspinal cord. The cytokine, once past the blood-brain barrier and in theCNS, can then be delivered to various areas of the brain or spinal cordthrough lymphatic channels, through a perivascular space, or transportthrough or along neurons. In one embodiment, the cytokine preferablyaccumulates in areas having the greatest density of receptor or bindingsites for that cytokine.

[0111] Use of a neural pathway to transport a cytokine to the lymphaticsystem, lacrimal gland, brain, spinal cord, or other components of thecentral nervous system obviates the obstacle presented by theblood-brain barrier so that medications that cannot normally cross thatbarrier, can be delivered directly to the brain, cerebellum, brain stem,or spinal cord. Although the cytokine that is administered may beabsorbed into the bloodstream as well as the neural pathway, thecytokine preferably provides minimal effects systemically. In addition,the invention can provide for delivery of a more concentrated level ofthe cytokine to neural cells since the cytokine does not become dilutedin fluids present in the bloodstream. As such, the invention provides animproved method for delivering a cytokine to the lymphatic system, CNS,brain, and/or spinal cord.

[0112] The Olfactory Neural Pathway

[0113] One embodiment of the present method includes delivery of thecytokine to the subject in a manner such that the cytokine istransported into the CNS, brain, and/or spinal cord along an olfactoryneural pathway. Typically, such an embodiment includes administering thecytokine to tissue innervated by the olfactory nerve and inside thenasal cavity. The olfactory neural pathway innervates primarily theolfactory epithelium in the upper third of the nasal cavity, asdescribed above. Application of the cytokine to a tissue innervated bythe olfactory nerve can deliver the cytokine to damaged neurons or cellsof the CNS, brain, and/or spinal cord. Olfactory neurons innervate thistissue and can provide a direct connection to the CNS, brain, and/orspinal cord due, it is believed, to their role in olfaction.

[0114] Delivery through the olfactory neural pathway can employlymphatics that travel with the olfactory nerve to the various brainareas and from there into dural lymphatics associated with portions ofthe CNS, such as the spinal cord. Transport along the olfactory nervecan also deliver cytokines to an olfactory bulb. A perivascular pathwayand/or a hemangiolymphatic pathway, such as lymphatic channels runningwithin the adventitia of cerebral blood vessels, can provide anadditional mechanism for transport of therapeutic cytokines to the brainand spinal cord from tissue innervated by the olfactory nerve.

[0115] A cytokine can be administered to the olfactory nerve, forexample, through the olfactory epithelium. Such administration canemploy extracellular or intracellular (e.g., transneuronal) anterogradeand retrograde transport of the cytokine entering through the olfactorynerves to the brain and its meninges, to the brain stem, or to thespinal cord. Once the cytokine is dispensed into or onto tissueinnervated by the olfactory nerve, the cytokine may transport throughthe tissue and travel along olfactory neurons into areas of the CNSincluding the brain stem, cerebellum, spinal cord, olfactory bulb, andcortical and subcortical structures.

[0116] Delivery through the olfactory neural pathway can employ movementof a cytokine into or across mucosa or epithelium into the olfactorynerve or into a lymphatic, a blood vessel perivascular space, a bloodvessel adventitia, or a blood vessel lymphatic that travels with theolfactory nerve to the brain and from there into meningial lymphaticsassociated with portions of the CNS such as the spinal cord. Bloodvessel lymphatics include lymphatic channels that are around the bloodvessels on the outside of the blood vessels. This also is referred to asthe hemangiolymphatic system. Introduction of a cytokine into the bloodvessel lymphatics does not necessarily introduce the cytokine into theblood.

[0117] The Trigeminal Neural Pathway

[0118] One embodiment of the present method includes delivery of thecytokine to the subject in a manner such that the cytokine istransported into the CNS, brain, and/or spinal cord along a trigeminalneural pathway. Typically, such an embodiment includes administering thecytokine to tissue innervated by the trigeminal nerve including insideand outside the nasal cavity. The trigeminal neural pathway innervatesvarious tissues of the head and face, as described above. In particular,the trigeminal nerve innervates the nasal, sinusoidal, oral andconjunctival mucosa or epithelium, and the skin of the face. Applicationof the cytokine to a tissue innervated by the trigeminal nerve candeliver the cytokine to damaged neurons or cells of the CNS, brain,and/or spinal cord to cells of the lymphatic system. Trigeminal neuronsinnervate these tissues and can provide a direct connection to the CNS,brain, and/or spinal cord due, it is believed, to their role in thecommon chemical sense including mechanical sensation, thermal sensationand nociception (for example detection of hot spices and of noxiouschemicals).

[0119] Delivery through the trigeminal neural pathway can employlymphatics that travel with the trigeminal nerve to the pons and otherbrain areas and from there into dural lymphatics associated withportions of the CNS, such as the spinal cord. Transport along thetrigeminal nerve can also deliver cytokines to an olfactory bulb. Aperivascular pathway and/or a hemangiolymphatic pathway, such aslymphatic channels running within the adventitia of cerebral bloodvessels, can provide an additional mechanism for transport oftherapeutic cytokines to the spinal cord from tissue innervated by thetrigeminal nerve.

[0120] The trigeminal nerve includes large diameter axons, which mediatemechanical sensation, e.g., touch, and small diameter axons, whichmediate pain and thermal sensation, both of whose cell bodies arelocated in the semilunar (or trigeminal) ganglion or the mesencephalictrigeminal nucleus in the midbrain. Certain portions of the trigeminalnerve extend into the nasal cavity, oral and conjunctival mucosa and/orepithelium. Other portions of the trigeminal nerve extend into the skinof the face, forehead, upper eyelid, lower eyelid, dorsum of the nose,side of the nose, upper lip, cheek, chin, scalp and teeth. Individualfibers of the trigeminal nerve collect into a large bundle, travelunderneath the brain and enter the ventral aspect of the pons. Acytokine can be administered to the trigeminal nerve, for example,through the nasal cavity's, oral, lingual, and/or conjunctival mucosaand/or epithelium; or through the skin of the face, forehead, uppereyelid, lower eyelid, dorsum of the nose, side of the nose, upper lip,cheek, chin, scalp and teeth. Such administration can employextracellular or intracellular (e.g., transneuronal) anterograde andretrograde transport of the cytokine entering through the trigeminalnerves to the brain and its meninges, to the brain stem, or to thespinal cord. Once the cytokine is dispensed into or onto tissueinnervated by the trigeminal nerve, the cytokine may transport throughthe tissue and travel along trigeminal neurons into areas of the CNSincluding the brain stem, cerebellum, spinal cord, olfactory bulb, andcortical and subcortical structures.

[0121] Delivery through the trigeminal neural pathway can employmovement of a cytokine across skin, mucosa, or epithelium into thetrigeminal nerve or into a lymphatic, a blood vessel perivascular space,a blood vessel adventitia, or a blood vessel lymphatic that travels withthe trigeminal nerve to the pons and from there into meningiallymphatics associated with portions of the CNS such as the spinal cord.Blood vessel lymphatics include lymphatic channels that are around theblood vessels on the outside of the blood vessels. This also is referredto as the hemangiolymphatic system. Introduction of a cytokine into theblood vessel lymphatics does not necessarily introduce the cytokine intothe blood.

[0122] Neural Pathways and Nasal Administration

[0123] In one embodiment, the method of the invention can employdelivery by a neural pathway, e.g., a trigeminal or olfactory neuralpathway, after administration to the nasal cavity. Upon administrationto the nasal cavity, delivery via the trigeminal neural pathway mayemploy movement of a cytokine through the nasal mucosa and/or epitheliumto reach a trigeminal nerve or a perivascular and/or lymphatic channelthat travels with the nerve. Upon administration to the nasal cavity,delivery via the olfactory neural pathway may employ movement of acytokine through the nasal mucosa and/or epithelium to reach theolfactory nerve or a perivascular and/or lymphatic channel that travelswith the nerve.

[0124] For example, the cytokine can be administered to the nasal cavityin a manner that employs extracellular or intracellular (e.g.,transneuronal) anterograde and retrograde transport into and along thetrigeminal and/or olfactory nerves to reach the brain, the brain stem,or the spinal cord. Once the cytokine is dispensed into or onto nasalmucosa and/or epithelium innervated by the trigeminal and/or olfactorynerve, the cytokine may transport through the nasal mucosa and/orepithelium and travel along trigeminal and/or olfactory neurons intoareas of the CNS including the brain stem, cerebellum, spinal cord,olfactory bulb, and cortical and subcortical structures. Alternatively,administration to the nasal cavity can result in delivery of a cytokineinto a blood vessel perivascular space or a lymphatic that travels withthe trigeminal and/or olfactory nerve to the pons, olfactory bulb, andother brain areas, and from there into meningeal lymphatics associatedwith portions of the CNS such as the spinal cord. Transport along thetrigeminal and/or olfactory nerve may also deliver cytokinesadministered to the nasal cavity to the olfactory bulb, midbrain,diencephalon, medulla, and cerebellum. A cytokine administered to thenasal cavity can enter the ventral dura of the brain and travel inlymphatic channels within the dura.

[0125] In addition, the method of the invention can be carried out in away that employs a perivascular pathway and/or an hemangiolymphaticpathway, such as a lymphatic channel running within the adventitia of acerebral blood vessel, to provide an additional mechanism for transportof cytokine to the spinal cord from the nasal mucosa and/or epithelium.A cytokine transported by the hemangiolymphatic pathway does notnecessarily enter the circulation. Blood vessel lymphatics associatedwith the circle of Willis as well as blood vessels following thetrigeminal and/or olfactory nerve can also be involved in the transportof the cytokine.

[0126] Administration to the nasal cavity employing a neural pathway candeliver a cytokine to the lymphatic system, brain stem, cerebellum,spinal cord, and cortical and subcortical structures. The cytokine alonemay facilitate this movement into the CNS, brain, and/or spinal cord.Alternatively, the carrier or other transfer-promoting factors mayassist in the transport of the cytokine into and along the trigeminaland/or olfactory neural pathway. Administration to the nasal cavity of atherapeutic cytokine can bypass the blood-brain barrier through atransport system from the nasal mucosa and/or epithelium to the brainand spinal cord.

[0127] Neural Pathways and Transdermal Administration

[0128] In one embodiment, the method of the invention can employdelivery by a neural pathway, e.g., a trigeminal neural pathway, aftertransdermal administration. Upon transdermal administration, deliveryvia the trigeminal neural pathway may employ movement of a cytokinethrough the skin to reach a trigeminal nerve or a perivascular and/orlymphatic channel that travels with the nerve.

[0129] For example, the cytokine can be administered transdermally in amanner that employs extracellular or intracellular (e.g., transneuronal)anterograde and retrograde transport into and along the trigeminalnerves to reach the brain, the brain stem, or the spinal cord. Once thecytokine is dispensed into or onto skin innervated by the trigeminalnerve, the cytokine may transport through the skin and travel alongtrigeminal neurons into areas of the CNS including the brain stem,cerebellum, spinal cord, olfactory bulb, and cortical and subcorticalstructures. Alternatively, transdermal administration can result indelivery of a cytokine into a blood vessel perivascular space or alymphatic that travels with the trigeminal nerve to the pons, olfactorybulb, and other brain areas, and from there into meningeal lymphaticsassociated with portions of the CNS such as the spinal cord. Transportalong the trigeminal nerve may also deliver transdermally administeredcytokines to the olfactory bulb, midbrain, diencephalon, medulla andcerebellum. The ethmoidal branch of the trigeminal nerve enters thecribriform region. An transdermally administered cytokine can enter theventral dura of the brain and travel in lymphatic channels within thedura.

[0130] In addition, the method of the invention can be carried out in away that employs a perivascular pathway and/or an hemangiolymphaticpathway, such as a lymphatic channel running within the adventitia of acerebral blood vessel, to provide an additional mechanism for transportof cytokine to the spinal cord from the skin. A cytokine transported bythe hemangiolymphatic pathway does not necessarily enter thecirculation. Blood vessel lymphatics associated with the circle ofWillis as well as blood vessels following the trigeminal nerve can alsobe involved in the transport of the cytokine.

[0131] Transdermal administration employing a neural pathway can delivera cytokine to the brain stem, cerebellum, spinal cord and cortical andsubcortical structures. The cytokine alone may facilitate this movementinto the CNS, brain, and/or spinal cord. Alternatively, the carrier orother transfer-promoting factors may assist in the transport of thecytokine into and along the trigeminal neural pathway. Transdermaladministration of a therapeutic cytokine can bypass the blood-brainbarrier through a transport system from the skin to the brain and spinalcord.

[0132] Neural Pathways and Sublingual Administration

[0133] In another embodiment, the method of the invention can employdelivery by a neural pathway, e.g., a trigeminal neural pathway, aftersublingual administration. Upon sublingual administration, delivery viathe trigeminal neural pathway may employ movement of a cytokine fromunder the tongue and across the lingual epithelium to reach a trigeminalnerve or a perivascular or lymphatic channel that travels with thenerve.

[0134] For example, the cytokine can be administered sublingually in amanner that employs extracellular or intracellular (e.g., transneuronal)anterograde and retrograde transport through the oral mucosa and theninto and along the trigeminal nerves to reach the brain, the brain stem,or the spinal cord. Once the cytokine is administered sublingually, thecytokine may transport through the oral mucosa by means of theperipheral processes of trigeminal neurons into areas of the CNSincluding the brain stem, spinal cord and cortical and subcorticalstructures. Alternatively, sublingual administration can result indelivery of a cytokine into lymphatics that travel with the trigeminalnerve to the pons and other brain areas and from there into meningeallymphatics associated with portions of the CNS such as the spinal cord.Transport along the trigeminal nerve may also deliver sublinguallyadministered cytokines to the olfactory bulbs, midbrain, diencephalon,medulla and cerebellum. The ethmoidal branch of the trigeminal nerveenters the cribriform region. A sublingually administered cytokine canenter the ventral dura of the brain and travel in lymphatic channelswithin the dura.

[0135] In addition, the method of the invention can be carried out in away that employs an hemangiolymphatic pathway, such as a lymphaticchannel running within the adventitia of a cerebral blood vessel, toprovide an additional mechanism for transport of a cytokine to thespinal cord from the oral submucosa. A cytokine transported by thehemangiolymphatic pathway does not necessarily enter the circulation.Blood vessel lymphatics associated with the circle of Willis as well asblood vessels following the trigeminal nerve can also be involved in thetransport of the cytokine.

[0136] Sublingual administration employing a neural pathway can delivera cytokine to the brain stem, cerebellum, spinal cord and cortical andsubcortical structures. The cytokine alone may facilitate this movementinto the CNS, brain, and/or spinal cord. Alternatively, the carrier orother transfer-promoting factors may assist in the transport of thecytokine into and along the trigeminal neural pathway. Sublingualadministration of a therapeutic cytokine can bypass the blood-brainbarrier through a transport system from the oral mucosa to the brain andspinal cord.

[0137] Neural Pathways and Conjunctival Administration

[0138] In another embodiment, the method of the invention can employdelivery by a neural pathway, e.g. a trigeminal neural pathway, afterconjunctival administration. Upon conjunctival administration, deliveryvia the trigeminal neural pathway may employ movement of a cytokine fromthe conjunctiva through the conjunctival epithelium to reach thetrigeminal nerves or lymphatic channels that travel with the nerve.

[0139] For example, the cytokine can be administered conjunctivally in amanner that employs extracellular or intracellular (e.g., transneuronal)anterograde and retrograde transport through the conjunctival mucosa andthen into and along the trigeminal nerves to reach the brain, the brainstem, or the spinal cord. Once the cytokine is administeredconjunctivally, the cytokine may transport through the conjunctivalmucosa by means of the peripheral processes of trigeminal neurons intoareas of the CNS including the brain stem, spinal cord and cortical andsubcortical structures. Alternatively, conjunctival administration canresult in delivery of a cytokine into lymphatics that travel with thetrigeminal nerve to the pons and other brain areas and from there intomeningeal lymphatics associated with portions of the CNS such as thespinal cord. Transport along the trigeminal nerve may also deliverconjunctivally administered cytokines to the olfactory bulbs, midbrain,diencephalon, medulla and cerebellum. The ethmoidal branch of thetrigeminal nerve enters the cribriform region. An conjunctivallyadministered cytokine can enter the ventral dura of the brain and travelin lymphatic channels within the dura.

[0140] In addition, the method of the invention can be carried out in away that employs an hemangiolymphatic pathway, such as a lymphaticchannel running within the adventitia of cerebral blood vessel, toprovide an additional mechanism for transport of a cytokine to thespinal cord from the conjunctival submucosa. A cytokine transported bythe hemangiolymphatic pathway does not necessarily enter thecirculation. Blood vessel lymphatics associated with the circle ofWillis as well as blood vessels following the trigeminal nerve can alsobe involved in the transport of the cytokine.

[0141] Conjunctival administration employing a neural pathway candeliver a cytokine to the brain stem, cerebellum, spinal cord andcortical and subcortical structures. The cytokine alone may facilitatethis movement into the CNS, brain, and/or spinal cord. Alternatively,the carrier or other transfer-promoting factors may assist in thetransport of the cytokine into and along the trigeminal neural pathway.Conjunctival administration of a therapeutic cytokine can bypass theblood-brain barrier through a transport system from the conjunctivalmucosa to the brain and spinal cord.

[0142] Articles and Methods of Manufacture

[0143] The present invention also includes an article of manufactureproviding a cytokine for administration to the CNS, brain, and/or spinalcord. The article of manufacture can include a vial or other containerthat contains a composition suitable for the present method togetherwith any carrier, either dried or in liquid form. The article ofmanufacture further includes instructions in the form of a label on thecontainer and/or in the form of an insert included in a box in which thecontainer is packaged, for the carrying out the method of the invention.The instructions can also be printed on the box in which the vial ispackaged. The instructions contain information such as sufficient dosageand administration information so as to allow the subject or a worker inthe field to administer the cytokine. It is anticipated that a worker inthe field encompasses any doctor, nurse, technician, spouse, or othercare-giver that might administer the cytokine. The cytokine can also beself-administered by the subject.

[0144] According to the invention, a cytokine can be used formanufacturing a cytokine composition or medicament suitable forintranasal, conjunctival, transdermal, and/or sublingual administration.For example, a liquid or solid composition can be manufactured inseveral ways, using conventional techniques. A liquid composition can bemanufactured by dissolving a cytokine in a suitable solvent, such aswater, at an appropriate pH, including buffers or other excipients, forexample to form a solution described herein above.

[0145] Disorders of the Central Nervous System

[0146] In one embodiment, the present method can be employed to delivercytokines, particularly IFN-β, to the brain for diagnosis, treatment orprevention of disorders or diseases of the CNS, brain, and/or spinalcord. IFN-β increases the astrocytic production of nerve growth factor(NGF) (Boutros et al (1997) Journal of Neurochemistry 69:939-946) andIFN-β sustains neuronal growth in cell culture (Plioplys et al. (1995)Neuroimmunodulation 2:131-135). IFN-β has therefore been associated withneurotrophic activity; hence, the methods of the present invention canbe used for the delivery of a cytokine to the CNS to treat or preventdisorders or diseases of the CNS, brain, and/or spinal cord.

[0147] Disorders of the CNS, brain and/or spinal cord can be neurologicor psychiatric disorders, and include, for example, brain diseases suchas Alzheimer's disease, Parkinson's disease, Lewy body dementia,multiple sclerosis, epilepsy, cerebellar ataxia, progressivesupranuclear palsy, amyotrophic lateral sclerosis, affective disorders,anxiety disorders, obsessive compulsive disorders, personalitydisorders, attention deficit disorder, attention deficit hyperactivitydisorder, Tourette Syndrome, Tay Sachs, Nieman Pick, and other lipidstorage and genetic brain diseases and/or schizophrenia. The method canalso be employed in subjects suffering from or at risk for nerve damagefrom cerebrovascular disorders such as stroke in the brain or spinalcord, from CNS infections including meningitis and HIV, from tumors ofthe brain and spinal cord, or from a prior disease. The method can alsobe employed to deliver cytokines to counter CNS disorders resulting fromordinary aging (e.g., anosmia or loss of the general chemical sense),brain injury, or spinal cord injury.

[0148] Multiple sclerosis is a preferred disease or disorder of the CNS,brain, and/or spinal cord. Despite its possible presence in theperiphery, multiple sclerosis is a disease of the CNS. Accordingly,multiple sclerosis may be targeted more efficiently by a methoddelivering interferons to the CNS, brain and/or spinal cord.

[0149] Another preferred disease of the CNS, brain, and/or spinal cordis meningitis.

[0150] An “effective amount” of a cytokine is an amount sufficient toprevent, treat, reduce and/or ameliorate the symptoms and/or underlyingcauses of any of the above disorders or diseases discussed herein. Insome instances, an “effective amount” is sufficient to eliminate thesymptoms of those diseases and, perhaps, overcome the disease itself. Inthe context of the present invention, the terms “treat” and “therapy”and the like refer to alleviate, slow the progression, prophylaxis,attenuation or cure of existing disease. Prevent, as used herein, refersto delaying, slowing, inhibiting, reducing or ameliorating the onset ofthe CNS or brain diseases or disorders. It is preferred that asufficient quantity of the cytokine be applied in non-toxic levels inorder to provide an effective level of activity within the CNS toprevent or treat the disease. The method of the present invention may beused with any mammal. Exemplary mammals include, but are not limited torats, cats, dogs, horses, cows, sheep, pigs, and more preferably humans.

[0151] Further Embodiments

[0152] Modulation of Immune and Inflammatory Responses

[0153] The method of cytokine administration provided by the presentinvention allows for the directed administration of the cytokine to thenasal lymphatic system. Following entry of the cytokine into the nasallymphatics, the cytokine can be distributed throughout the lymphatics ofthe head and neck region. Hence, the method of the present invention canbe employed to deliver cytokines to the lymphatic system including, forexample, the deep and superficial cervical nodes, and to various tissuesof the head and neck for the treatment or prevention of disorders ordiseases characterized by immune and inflammatory responses (i.e.,diseases resulting in acute or chronic inflammation and/or infiltrationby lymphocytes). As such the present invention provides a method tomodulate the immune response. By modulate is intended any up or downregulation of the immune or inflammatory response (i.e., influencingsystemic immune function, antigen presentation, cytokine production, andentry of leukocytes into the CNS).

[0154] Of particular interest in the methods of the invention is theadministration of IFN-β. IFN-β, like many of the interferons, reportedlyserves as an immunomodulator on a number of target cells (Hall et al.(1997) J Neuroimmunol. 72:11-19). For instance, IFN-β appears to exertantiproliferative action on macrophages, counteract “the mitogenicstimulus of certain cytokines”, augment natural killer cell activity toinduce an increase in the production of cytotoxic T lymphocytes, and acton large, granular lymphocytes to increase killer cell activity.Additionally, IFN-β augments the proliferation of B cells and thesecretion of IgM, IgG, and IgA. It has been shown to upregulate class IMHC expression to produce an increase in the presentation of class Irestricted antigen CD8 cells (Hall et al. (1997) J NeuroimmunoL72:11-19). Conversely, IFN-β exerts an inhibitory effect on theupregulation of class II surface expression. Hence, the immunomodulatoryactivities of IFN-β include, for example, influencing systemic immunefunction, antigen presentation, cytokine production, and entry ofleukocytes into the CNS (Yong et al. (1998) Neurology 51:582-689).Direct delivery of the cytokine to the lymphatics of the head and neckusing the administration methods of the present invention allows thecytokine to modulate the immune response, i.e., influence chronic andacute inflammation, wound healing, and the autoimmune response; modulatethe function by lymphocytes (reduce lymphocyte infiltration of theinjured tissue); etc.

[0155] Given the immunomodulatory role of cytokines, the presentinvention can be employed to deliver cytokines, preferably IFN-β, tovarious tissues of the head and neck for the treatment and/or preventionof diseases or disorders characterized by immune and inflammatoryresponses. Disorders or diseases of particular interest include MultipleSclerosis (MS), meningitis, and Primary Sjogren's Syndrome.

[0156] MS presents in the white matter of the CNS and spinal cord as anumber of sclerotic lesions or plaques (Prineas (1985) DemyelinatingDiseases, Elsvevier: Amsterdam; Raine (1983) Multiple Sclerosis,Williams and Wilkins: Baltimore; Raine et al. (1988) J. Neuroimmunol.20:189-201; and Martin (1997) J. Neural Transmission (Suppl) 49:53-67).The characteristic MS lesion is inflamed, exhibits axonal demyelination,axonal degeneration, and is found around small venules. Thesecharacteristics typically evolve early in plaque development and arehypothesized to occur as a result of a breakdown in the blood-brainbarrier (BBB). As a consequence of BBB breakdown, infiltrates consistingof various lymphocytes and macrophages enter the brain. The infiltratescause a decrease in inflammation while increasing the presence of glialscar tissue, and elicit incomplete remyelination (Martin (1997) J.Neural Transmission (Suppl) 49:53-67). Further, it is hypothesized thatthis apparent immunologic attack targets not only the myelin sheath, butalso the oligodendrocytes imperative to CNS myelin production. Cytokinesare known to effectively reduce the symptoms of MS. For example,interferon-β (IFN-β) has received interest as a treatment forrelapsing-remitting MS. In addition, interest has also developed in theuse of interferon-i as an effective treatment in autoimmune diseases,such as MS. See, for example, U.S. Pat. No. 6,060,450, hereinincorporated by reference.

[0157] The immunomodulating activity of IFN-β influences the clinicalsymptoms of MS. Hence, IFN-β can be administered according to themethods of the present invention to treat MS. While the presentinvention is not bound by the mechanism of IFN-β action, the centralnervous system damage that ensues in MS patients is believed to be dueto the delayed-type hypersensitivity response. This is a cell-mediatedresponse. First, T cells are activated by antigens and conveyed to thelymphoid organ (activation). The lymphoid organ then activates these Tcells while continuing to recruit more T cells to its site(recruitment). The activated lymphocytes proliferate and return tocirculation (expansion). Once returned to circulation, the activatedlymphocytes migrate through the blood stream, crossing endothelial cellslining the capillaries (migration). These migrating lymphocytes andmacrophages target, and are attracted to the area of inflammation(attraction). Resulting from this attraction, other lymphocytes continueto the area of inflammation and tissue is destroyed (tissuedestruction). Subsequently, the acute response is suppressed (via tissuedestruction), and repair of the area of inflammation, which is quitelimited in MS, may commence (repair) (Kelley (1996) J. of NeuroscienceNursing 28:114-120). Therefore, the migration of activated lymphocytesfrom the blood initiates the immune response, thereby allowing BBBpenetration of activated lymphocytes.

[0158] Evidence suggests that the immunomodulatory activity of IFN-βinhibits IFN-γ upregulation by inhibiting the expansion stage of thedelayed-type hypersensitivity response and thereby influences theclinical symptoms of MS. Particularly, the reduction of myelin damageappears to occur as a result of two hypothesized mechanisms of IFN-βaction: (1) inhibition of IFN-γ-induced macrophage activation, and (2)inhibition of monocytotic TNF release (Kelly (1996) J. NeuroscienceNursing 28:114-120). Potential sites of IFN-β action construed by thesehypotheses involve systemic immune function, antigen presentation,cytokine production, and entry of leukocytes into the CNS (Yong et al.(1998) Neurology 51:682-689). Each of these sites has been elaborated inhuman and animal experiments of MS.

[0159] An “effective amount” of a cytokine to treat MS using theadministration methods of the present invention will be sufficient toreduce or lessen the clinical symptoms of MS. For instance, experimentalallergic encephalomyelitis (EAE) is commonly used as an animal model ofMS. A therapeutically effect amount of a cytokine delivered by themethods of the present invention will be such as to improve the clinicalsymptoms of EAE in the experimental animal (i.e., rats or mice). EAE inrats is scored on a scale of 0-4:0, clinically normal; 1, flaccid tailparalysis; 2, hind limb weakness; 3, hind limb paralysis; 4, front andhind limb affected. An effective amount of cytokine delivered by themethods of the present invention will be effective if there is at leasta 30%, 40%, 50% or greater reduction in the mean cumulative score overseveral days following the onset of disease symptoms in comparison tothe control group.

[0160] Furthermore, effective treatment of MS may be examined in severalalternative ways including, EDSS (extended disability status scale),appearance of exacerbations, or MRI. Satisfying any of the followingcriteria evidences effective treatment.

[0161] The EDSS is a means to grade clinical impairment due to MS(Kurtzke (1983) Neurology 33:1444). Eight functional systems areevaluated for the type and severity of neurologic impairment. Briefly,prior to treatment, impairment in the following systems is evaluated:pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual,cerebral, and other. Follow-ups are conducted at defined intervals. Thescale ranges from 0 (normal) to 10 (death due to MS). A decrease of onefull step defines an effective treatment in the context of the presentinvention (Kurtzke (1994) Ann. NeuroL 36:573-79).

[0162] Exacerbations are defined as the appearance of a new symptom thatis attributable to MS and accompanied by an appropriate new neurologicabnormality (IFN-β MS Study Group, supra). In addition, the exacerbationmust last at least 24 hours and be preceded by stability or improvementfor at least 30 days. Standard neurological examinations result in theexacerbations being classified as either mild, moderate, or severeaccording to changes in aNeurological Rating Scale (Sipe et al. (1984)Neurology 34:1368). An annual exacerbation rate and proportion ofexacerbation-free patients are determined. Therapy is deemed to beeffective if there is a statistically significant difference in the rateor proportion of exacerbation-free patients between the treated groupand the placebo group for either of these measurements. In addition,time to first exacerbation and exacerbation duration and severity mayalso be measured. A measure of effectiveness as therapy in this regardis a statistically significant difference in the time to firstexacerbation or duration and severity in the treated group compared tocontrol group.

[0163] MRI can be used to measure active lesions usinggadolinium-DTPA-enhanced imaging (McDonald et al (1994) Ann. Neurol.36:14) or the location and extent of lesions using T₂-weightedtechniques. Briefly, baseline MRIs are obtained. The same imaging planeand patient position are used for each subsequent study. Areas oflesions are outlined and summed slice by slice for total lesion area.Three analyses may be done: evidence of new lesions, rate of appearanceof active lesions, and percentage change in lesion area (Paty et al.(1993) Neurology 43:665). Improvement due to therapy is established whenthere is a statistically significant improvement in an individualpatient compared to baseline or in a treated group versus a placebogroup.

[0164] It is further recognized that additional compounds can beadministered with the cytokine to produce a therapeutic effect. Forinstance, IGF-1 has been implicated in preventing the depletion ofmature oligodendrocytes and promoting recovery from demyelination in MSand other demyelinating disorders. See, for example, Mason et al. (2000)J. Neuroscience 20:5703-5708, herein incorporated by reference. Hence,IFN-β can be administered in conjunction with IGF-1 for the treatment ofMS. The compounds can be administered by the methods of the invention.Alternatively, one of the compounds can be administered by any methodknown in the art including, for example, subcutaneous and intramuscularroutes.

[0165] The IGF-1 used according to the methods of the present inventioncan be in its substantially purified, native, recombinantly produced, orchemically synthesized forms. For example, IGF-1 can be isolateddirectly from blood, such as from serum or plasma, by known methods.See, for example, Phillips (1980) New Eng. J Med. 302:371-380; Svobodaet al. (1980) Biochemistry 19:790-797; Cornell and Boughdady (1982)Prep. Biochem. 12:57; Cornell and Boughdady (1984) Prep. Biochem.14:123; European Patent No. EP 123,228; and U.S. Pat. No. 4,769,361.IGF-1 may also be recombinantly produced in the yeast strain Pichiapastoris and purified essentially as described in U.S. Pat. Nos.5,324,639, 5,324,660, and 5,650,496 and International Publication No. WO96/40776; all of which are herein incorporated by reference.

[0166] Alternatively, IGF-1 can be synthesized chemically, by any ofseveral techniques that are known to those skilled in the peptide art.See, for example, Li et al. (1983) Proc. Natl. Acad. Sci. USA80:2216-2220, Stewart and Young (1984) Solid Phase Peptide Synthesis(Pierce Chemical Company, Rockford, Ill.), and Barany and Merrifield(1980) The Peptides: Analysis, Synthesis, Biology, ed. Gross andMeienhofer, Vol. 2 (Academic Press, New York, 1980), pp. 3-254, forsolid phase peptide synthesis techniques; and Bodansky (1984) Principlesof Peptide Synthesis (Springer-Verlag, Berlin); and Gross andMeienhofer, eds. (1980) The Peptides: Analysis, Synthesis, Biology, Vol.1 (Academic Press, New York), for classical solution synthesis. IGF-1can also be chemically prepared by the method of simultaneous multiplepeptide synthesis. See, for example, Houghten (1985) Proc. Natl. Acad.Sci. USA 82:5131-5135; and U.S. Pat. No. 4,631,211. These references areherein incorporated by reference. Furthermore, methods to prepare ahighly concentrated, low salt-containing, biologically active form ofIGF-1 or variant thereof are provided in WO 99/24062, entitled NovelIGF-1 Compositions and Its Use.

[0167] Methods for making IGF-1 fragments, analogues, and derivativesare available in the art. See generally U.S. Pat. Nos. 4,738,921,5,158,875, and 5,077,276; International Publication Nos. WO 85/00831, WO92/04363, WO 87/01038, and WO 89/05822; and European Patent Nos. EP135094, EP 123228, and EP 128733; herein incorporated by reference.

[0168] In addition, several IGF-1 variants are known in the art andinclude those described in, for example, Proc. Natl. Acad. Sci. USA 83(1986):4904-4907; Biochem. Biophys. Res. Commun. 149 (1987):398-404; JBiol. Chem. 263 (1988):6233-6239; Biochem. Biophys. Res. Commun. 165(1989):766-771; Forsbert et al. (1990) Biochem. J. 271:357-363; U.S.Pat. Nos. 4,876,242 and 5,077,276; and International Publication Nos. WO87/01038 and WO 89/05822. Representative variants include one with adeletion of Glu-3 of the mature molecule, a variant with up to 5 aminoacids truncated from the N-terminus, a variant with a truncation of thefirst 3 N-terminal amino acids (referred to as des(1-3)-IGF-1,des-IGF-1, tIGF-1, or brain IGF), and a variant including the first 17amino acids of the B chain of human insulin in place of the first 16amino acids of human IGF-1.

[0169] Meningitis refers to an inflammatory process of the leptomeningesand CSF within the subarachnoid space. Meningoencephalitis applies toinflammation of the meninges and brain parenchyma. Meningitis is usuallycaused by an infection, but chemical meningitis may also occur inresponse to a non-bacterial irritant introduced into the subarachonoidspace. Infiltration of the subarachnoid space by carcinoma is referredto as meningeal carcinomatosis and by lymphoma as lymphomapyogenic(usually bacterial), aseptic (usually viral), and chronic (most anyinfectious agent).

[0170] It has been suggested that the central nervous system damage thatoccurs in viral and bacterial meningitis may be more related to invasionof the surface of the brain by the host's own lymphocytes in response tothe meningitis pathogen, rather than to the pathogen itself or any toxinproduced by the pathogen (Lewis (1979) The Medusa and The Snail, PenguinBooks). In fact, many patients fall victim to the disease despite theprompt sterilization of the cerebrospinal fluid using the currentaggressive treatments, such as the third generation cephalosporins. Thisunexpected outcome may result from harmful interactions between hostcells/tissues and bacterial components released by treatment with lyticantibiotics (Scand et al. (1991) J Infect., Dis. Supp. 74:173-179). Theburst of peptidoglycan, capsular polysaccharide, and lipopolysaccharideliberated from the bacteria induce the production of a number ofmediators including TNF in the central nervous system leading tomeningeal and perivascular inflammation in the subarachnoid space.Disruption of the blood-brain barrier ensues, leading to cerebral edema,ischemia, and a dramatic increase in intracranial pressure. Those thatsurvive the acute phase of the disease are often left with multipleneurological sequelae. Previous results from trials utilizingsteroid-based anti-inflammatories either prior to or concomitant withantibiotic administration suggest that such an approach may have value.See, for example, Mustafa et al. (1990) Amer. J. Diseases of Children144:883-887. Hence, administration of a cytokine, particularlyinterferon-β, using the methods of the present invention could beeffective in preventing damage by activated lymphocytes. The methods ofthe invention could be used in conjunction with the existing treatmentsfor meningitis to help prevent brain damage. Such treatments aredescribed in Harrison's Principles of Internal Medicine (McGraw Hill,1994), pp. 2296-2309, herein incorporated by reference.

[0171] An “effective amount” of a cytokine to treat meningitis using theadministration method of the present invention will be sufficient toreduce or lessen the clinical symptoms of meningitis. In preferredembodiments, the cytokine is administered in conjunction with anantibiotic regiment. As such, an effective amount of the cytokineaugments the activity of the antibiotics and leads to enhanced survivaland/or improved clinical status of the animals in comparison to animalstreated with antibiotics alone. Such clinical manifestations mayinclude, for example, 1) a more rapid normalization of the CNSinflammatory indices compared to a control; 2) a more rapiddisappearance in fever as compared to a control; 3) a reduction in theoverall neurologic sequelae; and/or, 4) an improved mortality ascompared to a control. More extensive details regarding the clinicalmanifestations of meningitis that can be improved upon theadministration of an effective concentration of a cytokine can be foundin Harrison's Principles of Internal Medicine (McGraw Hill, 1994), pp.2296-2309, herein incorporated by reference.

[0172] Primary Sjogren's Syndrome, also known as Dry Eye Syndrome, ischaracterized by decreased secretion of the lacrimal glands that makethe aqueous layer of the tear film that lubricates the eyes. Manypatients afflicted with Sjogren's Syndrome also experience dry mouth dueto decreased secretion of the salivary glands. This is an autoimmunedisease characterized by chronic inflammation and infiltration of thelacrimal and salivary glands by lymphocytes. Activated T cells of theCD4⁺ type that infiltrate the lacrimal gland mediate tissue destruction(Tabbara et al. (1999) Eur. J. Ophthalomol. 9:1-7). Recently,nHu-IFN-alpha administered by the oral mucosa route has been shown tostimulate output (Ship et al. (1999) J. Interferon Cytokine Res.19:480-488).

[0173] Hence, the present invention provides a method of administeringcytokines, particularly, IFN-α and IFN-β, such that the compoundsdirectly enter the nasal lymphatic system. The interferon will then bedistributed to the lymphatics of the head and neck region altering thefunction of the lymphocytes that affect the lacrimal and salivaryglands. It is further recognized that delivery of the cytokine via thetrigeminal or the olfactory nerve can result in the direct delivery ofthe cytokine to the lacrimal gland. This direct delivery of theinterferon to the lymphatics of the head and neck region or directly tothe lacrimal gland will reduce lymphocyte infiltration of the lacrimaland salivary glands and treat Sjogren's Syndrome.

[0174] An “effective amount” of a cytokine to treat Sjogren's Syndromeusing the administration method of the present invention will besufficient to reduce or lessen the clinical symptoms of Sjogren'sSyndrome. As such, an effective amount of the cytokine leads to animproved clinical status of a patient suffering from Sjogren's Syndromein comparison to an untreated patient. For instance, an improvedclinical status of the oral symptoms of Sjogren's Syndrome includes, forexample, an overall increase in mouth wetness, an improvement in theability to swallow dry food, an improvement in the ability to speakcontinuously, etc. Further, an effective concentration encompasses anyimprovement in the ocular manifestations of Sjogren's Syndromeincluding, for example, increase in the wetness of eyes (i.e., alessening of the sandy or gritty feeling under the eyelids), an increasein tearing, and a decrease in burning sensations, redness, itching, andeye fatigue. Improvements also encompass an improvement in lacrimalfunction (i.e., a reduction in lymphocyte infiltration into the lacrimalgland). A more extensive description of the clinical manifestation ofSjogren's Syndrome can be found in Harrison's Principles of InternalMedicine (McGraw Hill, 1994), pp. 1662-1664, herein incorporated byreference.

[0175] Treatment of Viral Infections

[0176] In another embodiment, the present method can be employed todeliver cytokines and/or antiviral agents to the lymphatic system, CNS,brain, and/or spinal cord for the treatment, diagnosis or prevention ofdisorders or disease resulting from viral infection.

[0177] As used herein “treating or preventing viral infection” means toinhibit virus transmission, or to prevent the virus from establishingitself in its host CNS, brain or spinal cord, or to ameliorate oralleviate the symptoms of the disease caused by viral infection. Thetreatment is considered therapeutic if there is a reduction in viralload in the CNS, brain, or spinal cord, decrease in mortality, and/ormorbidity. Of particular interest is the administration of a cytokine(particularly IFN-α or IFN-β) by the methods of the invention for thetreatment or prevention of viral hepatitis.

[0178] Viral hepatitis refers to an infection of the liver caused by agroup of viruses having a particular affinity for the liver and includehepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis Dvirus, and hepatitis E virus. Of particular interest is the use of thepresent invention for the treatment of hepatitis C.

[0179] Acute infection with hepatitis C virus results in persistentviral replication and progression to chronic hepatitis in approximately90% of cases. While chronic hepatitis C infection is commonly treatedwith IFN-β and IFN-α, less than 50% of the patients have sustainedremission following treatment (i.e., the eradication of hepatitis Cvirus). See, for example, Barbaro et al. (1999) Scand. J. Gastroenterol.9:928-933; Oketani et al. (1999) J. Clin. Gastroenterol. 28:49-51; and,Kakizaki et al. (1999) J. Viral Hepatitis 6:315-319; all of which areherein incorporated by reference. Similarly, IFN therapy has also beendemonstrated to be an effective treatment for chronic hepatitis B,however only 25-40% of the patients profit from a long-term beneficialresponse to the current interferon therapies. Combination therapies forviral hepatitis have also been developed, which combine IFN-therapy withantiviral agents such as ribavirin. These IFN/antiviral therapies areusually given systemically (i.e., intravenously), and hence, thetherapeutic agents are not able to cross the blood-brain barrier. Thus,the hepatitis virus can harbor in the central nervous system where thetherapeutic agents cannot penetrate. Re-infection and relapse to viralhepatitis symptoms subsequent to treatment frequently occurs. Inaddition, viral hepatitis infection of the CNS can have seriousneurologic consequences. See, for example, Bolay et al. (1996) Clin.Neurol. Neurosurg. 98:305-308, herein incorporated by reference.Therefore, new methods of treatment are necessary in the treatment ofviral hepatitis. The methods of the present invention can be used toadminister a cytokine and/or an antiviral agent or any combinationthereof, to the lymphatic system, CNS, brain and/or spinal cord for thetreatment or prevention of viral hepatitis. The methods of the inventioncan be used in conjunction with the existing treatments for viralhepatitis to aid in reducing the clinical symptoms of hepatitis.

[0180] As used herein, an “effective amount” of a cytokine or anantiviral agent for the treatment of viral hepatitis using theadministration method of the present invention will be sufficient toreduce or lessen the clinical symptoms of hepatitis. As such, aneffective amount of the cytokine or antiviral agent administered by themethods of the present invention will augment the activity of thesystemically administered antiviral/immunomodulatory compounds used inthe art for the treatment of viral hepatitis. As such, the methods ofthe invention enhance survival and/or improve clinical status of thetreated animals in comparison to animals treated with systemicadministration methods alone. Improvement in clinical status includes,for example, the prevention of the progression of acute viral hepatitisto chronicity, the reduction of the viral load in chronic hepatitis,and/or the prevention or reduction in the frequency of re-infection andrelapse of viral hepatitis symptoms, and/or prevent or reduce theneurologic damage resulting from the viral infection.

[0181] Antiviral agents and cytokines of particular interest include,for example, ribavirin, thymosins, and cytokines, such as, IFN-β, IFN-α,and IFN-γ. See, for example, Musch et al. (1998) Hepato-Gastroenterology45:2282-2294; Barbaro et al. (1999) Scand. J. Gastroenterol.9(34):928-933; Oketani et al. (1999) J. Clin. Gastroenterol. 28:49-51;Kakizaki et al. (1999) J. Viral Hepatitis 6:315-319; U.S. Pat. No.6,030,785; U.S. Pat. No. 5,676,942; and U.S. Pat. No. 6,001,799; all ofwhich are herein incorporated by reference.

[0182] The course of the viral hepatitis and its response to thetreatments administered by the methods of the present invention may befollowed by clinical examination and laboratory findings that arecommonly performed in the art. For instance, elevated serum alanineaminotransferase (ALT) and aspartate aminotransferase (AST) are known tooccur in uncontrolled hepatitis C. A complete response to treatment isgenerally defined as the normalization of these serum enzymes,particularly ALT (Davis et al. (1989) New England J. Med. 321:1501-6).Alternatively, hepatitis C virus replication in subjects in response tothe antiviral/immunomodulatory treatment of the present invention can befollowed by measuring hepatitis C virus RNA in serum samples by, forexample, a nested polymerase chain reaction assay that uses two sets ofprimers derived from the NS3 and NS4 non-structural gene regions of theHCV genome (Farci et al. (1991) New England J. Med. 325:98-104; Ulrichet al. (1990) J. Clin. Invest. 86:1609-14).

[0183] In another embodiment, the methods of the present invention canbe used to treat or prevent herpes simplex viral infection. Herpessimplex viruses (HSV-1 and HSV-2) produce a variety of infectionsinvolving mucocutaneous surfaces, the central nervous system, andoccasionally visceral organs. For instance, acute viral replication at aperipheral site such as the cornea is followed by viral entry intoneuronal termini. Corneal infection is followed by intra-axonaltransport, which moves the virus to the trigeminal ganglia, wherefurther replication may occur before clearance of infectious virus andthe establishment of latency. Failure to clear the virus may result incentral nervous system infection, encephalitis, and death. Latency mayperiodically break down in response to certain stimuli, leading to viralreactivation and shedding. The present invention provides a method ofadministering a cytokine (via, for example, the trigeminal or olfactorynerve) to the trigeminal ganglia and/or the CNS, thereby allowing forthe treatment and/or prevention of herpes simplex viral infection.

[0184] The immune response to acute herpes simplex virus infectioninvolves both innate and acquired immunity. Key mediators of innateresistance to viral infection include cytokines, particularlyinterferons such as IFN-α, IFN-β, and IFN-γ. For instance, IFN-α hasbeen shown to inhibit the onset of immediate-early herpes simplex virusgene expression (Oberman et al. (1988) J. Gen. Virol. 69:1167-1177).Furthermore, in mice IFN-α and IFN-β are potent inhibitors ofreplication in the cornea. Specifically, studies have shown thatfollowing corneal inoculation in mice, herpes simplex viral titer inboth the eyes and trigeminal ganglia was enhanced by up to 1000 fold inmice mutant for IFN-α or IFN-β compared to wild-type control mice (Leibet al. (1999) J. Exp. Med. 189:663-672, herein incorporated byreference). The same study further demonstrated that IFNs significantlyreduce productive viral infection and reduce the spread of virus fromintact corneas. Related studies have also been preformed by Minagawa etal (1997) Antiviral Res. 36:99-105.

[0185] In addition, IFN-α and IFN-β activate host defenses such asnatural killer cells, which have themselves been shown to be importantin controlling herpes simplex virus infection and pathology (Bouley etal. (1996) Clin. Immunol. Immunopathol. 80:23-30). IFN-α and IFN-β havealso been suggested to be important for limiting progress of infectionfrom peripheral tissues to the nervous system (Halford et al. (1997)Virology 236:328-337). Furthermore, IFN-γ appears to play an importantrole in the clearance of herpes simplex virus from the cornea and inresistance to encephalitis, possibly by inhibiting apoptosis of neurons(Bouley et al. (1995) J. Immunol. 155:3964-3971, Geiger et al. (1997)Virology 238:189-197, and Imanishi et al. (2000) J. Biochem.127:525-530). Hence, interferons, particularly IFN-α, IFN-β, and IFN-γ,play a major role in limiting herpes simplex viral replication in thecornea, trigiminal ganglia, and in the nervous system.

[0186] An “effective amount” of a cytokine for the treatment of herpessimplex virus using the administration method of the present inventionwill be sufficient to reduce or lessen the clinical symptoms of herpessimplex virus. As such, an effective amount of the cytokine administeredby the methods of the present invention will attenuate the activity ofthe virus and thereby enhance survival and/or improve clinical status ofthe treated animal in comparison to the untreated control. Improvementin clinical status includes, for example, the prevention or reduction ofencephalitis and/or apoptosis in the central nervous system (i.e,increase in neuroprotection), a decrease in the severity of infection(i.e., enhancing viral clearance from the cornea, the trigeminalganglia, and the CNS), a decrease in viral spread, an increase in themaintenance of latency, and/or a decrease in the frequency of herpessimplex recurrences. More extensive details regarding the clinicalmanifestations of herpes simplex that can be improved upon theadministration of an effective concentration of a cytokine can be foundin Harrison's Principles of Internal Medicine (McGraw Hill, 1994), pp.782-787, herein incorporated by reference.

[0187] In another embodiment, the methods of the invention can be usedfor the treatment of human immunodeficiency virus (HIV). HIV is aninfectious disease of the immune system characterized by a progressivedeterioration of the immune system in most infected subjects. Duringdisease progression, key cells associated with the immune system becomeinfected with HIV, including, e.g., CD4⁺ T cells, macrophages/monocytes,and glial cells. Prolonged HIV infection frequently culminates in thedevelopment of AIDS. In the late stages of this disease, the immunesystem is severely compromised due to loss or dysfunction of CD4⁺ Tcells (Shearer et al. (1991) AIDS 5:245-253). The nervous system is alsoa major target of HIV infection. The virus is carried to the brain byinfected monocytes and the neurologic manifestations of HIV infectionare thought to arise from viral products and soluble factors produced bythe infected macrophages/microglia. Thus, the HIV virus can harbor inthe central nervous system where the therapeutic agents cannotpenetrate. Re-infection and relapse to HIV symptoms subsequent totreatment frequently occurs. Accordingly, the present invention providesa method of administering a cytokine, particularly an interferon such asIFN-α, IFN-β, and IFN-γ, to the CNS or the lymphatic system for thetreatment or prevention of HIV infection.

[0188] Interferons are known to exert pleiotropic antiretroviralactivities and affect many different stages of the HIV infectious cycle.For instance, IFN-β influences uptake of HIV particles (Vieillard et aL(1994) Proc. Natl. Acad. Sci. USA 91:2689-2693); reverse transcriptionof viral genomic RNA into proviral DNA (Baca-Regen et al. (1994) J.Virol. 68:7559-7565: Kombluth et al. (1990) Clin. Immunol. Immunopathol.54:200-219 and Shirazi et al. (1993) Virology 193:303-312); viralprotein synthesis (Coccia et aL (1994) J. Biol Chem. 269:23087-23094);and packaging and release of viral particles (Poli et al. (1989) Science244:575-577). In addition, virions released from IFN-β treated cells areup to 1,000-fold less infectious than equal numbers of virions releasedfrom untreated cells (Hansen et al. (1992) J. Virol. 66:7543-7548).Furthermore, recent studies have shown that genetically engineered humanCD4⁺ T cells producing constitutively low amounts of IFN-β can eradicateHIV in vivo using a mouse animal model that supports persistent,replicative HIV infection. These results indicated that a therapeuticstrategy based upon IFN-β transduction of CD4⁺ T cells may be successfulin controlling a preexisting HIV infection and allowing immunerestoration. See, for example, Vieillard et aL (1999) J. Virol.73:10281-10288, herein incorporated by reference. IFN-γ has also beenshown to modulate the susceptibility of macrophages to HIV (Zaitseva etal. (2000) Blood 96:3109-3117).

[0189] It is recognized that administration of the cytokine via themethods of the present invention for the treatment of HIV can be used incombination with any other HIV treatment or therapy known in the art.Therapies used in the treatment of HIV infection include, for example,anti-retroviral drugs, such as reverse transcriptase inhibitors, viralprotease inhibitors, and viral entry inhibitors (Caliendo et aL (1994)Clin. Infect. Dis. 18:516-524). More recently, treatment withcombinations of these agents, known as highly active antiretroviraltherapy (HAART), has been used to effectively suppress replication ofHIV (Gulick et al. (1997) N. Engl. J. Med. 337:734-9 and Hammer et al.(1997) N. Engl. J. Med. 337:725-733).

[0190] An “effective amount” of a cytokine for the treatment of HIVusing the administration method of the present invention will besufficient to reduce or lessen the clinical symptoms of HIV. As such, aneffective amount of the cytokine administered by the methods of thepresent invention will attenuate the activity of the virus (i.e., have adirect antiviral effect) and/or improve the HIV-induced immunologicaldysfuntions (i.e., enhance the ability of an HIV-infected patient toeffectively mount a cellular immune defense against actively replicatingHIV). Regardless of the mechanism of action, an effective amount of acytokine will enhance survival and/or improve clinical status of thetreated animals in comparison to the untreated control. Improvement inclinical status includes, for example, a reduction in preexisting HIVinfection and/or the rate of disease progression; enhanced CD4⁺ T-cellsurvival; suppression of cytokine dysregulation caused by HIV (i.e.,enhanced Th1-like cytokine expression); inhibition of viral replication;and improvement in the proliferative expansion of antigen-selectedlymphocytes, more particularly the HIV antigen-specific CD8⁺ subset of Tcells, in response to an increase in viral load. Assays to measure thesevarious improvements are known in the art. See, for example, Vieillardet al. (1999) J. Virol. 73:10281-10288, Vieillard et al. (1997) Proc.Natl. Acad. Sci. USA 94:11595-11600; U.S. Pat. No. 5,911,990 and U.S.Pat. No. 5,681,831; all of which are herein incorporated by reference.More extensive details regarding the clinical manifestations of HIV thatcan be improved upon the administration of an effective concentration ofa cytokine can be found in Harrison's Principles of Internal Medicine(McGraw Hill, 1994), pp. 1559-1617, herein incorporated by reference.

[0191] Treatment of Proliferative Disorders of the CNS

[0192] In another embodiment, the present method can be employed todeliver cytokines to the lymphatic system, CNS, brain, and/or spinalcord for the treatment, diagnosis or prevention of a proliferationdisorder or disease.

[0193] Cytokines have anti-proliferative activity. For instance,interferons have been shown to have both a direct cytotoxic effect ontumor cells and an indirect cytotoxic effect through the activation ofnatural killer cells, macrophages, or other immune cells. Specifically,studies have suggested IFN-γ mediated anti-tumor activity results frommodulating the interplay of B and T cell components of the immunesystem, as well as the inhibition of tumor angiogenesis (Saleh et al.(2000) Gene Ther 7:1715-24). IFN-α has also been shown to significantlydecrease average tumor size and increase the average survival time ofthe treated mammal (Wang et al. (1999) J Neuropathol Exp. Neurol58:847-58). Intratumoral injection of liposomes containing the humanIFN-β gene in nude mice inhibits tumor growth, with complete tumorregression occurring following multiple Intratumoral injections of thegene. Furthermore, IFN-β has been demonstrated to be an effectivetreatment of high grade astrocytomas (Natsume et al. (1999) Gene Ther.9:1626-33 and Fine et al. (1997) Clin. Cancer Res 3: 381-7). Theantiproliferative effect of IFN-β appears to occurs through an arrest inthe ordered progression through S phase or decreasing the entry intoG2/M phase of the cell cycle (Garrison et al. (1996) J Neurooncol30:213-23). Hence, interferons, particularly IFN-α, IFN-β, and IFN-γ,are effective agents for the treatment or prevention of a proliferationdisorder of the CNS, spinal cord, brain and lymphatic system.

[0194] By “proliferation disorder” is intended any disordercharacterized by cellular division occurring in defiance of the normaltissue homeostasis mechanism. The proliferation disorder can be eithermalignant or benign and result from either an increase in the rate ofcell proliferation or a decrease in the rate of cell death. Theproliferative disorder treated by the methods of the invention may be atany stage of development (i.e., an early stage with minimal ormicroscopic tumor burdens or at advanced stages of tumor development).

[0195] Proliferative disorders of the central nervous system, brain, orspinal cord include, for example, gliomas, neuronal tumors, poorlydifferentiated neoplasms, and meningiomas. Gliomas derived from glialcells include astrocytomas (i.e., fibrillary astrocytomas, glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthastrocytoma, andbrain stem glioma), oligodendrogliomas, and ependymomas andparaventricular mass lesions (i.e., myxopapillary ependymomas,subependymomas, choroid plexus papillomas). Neural tumors comprise CNStumors that contain mature-appearing neurons (ganglion cells) that mayconstitute the entire cell population of the lesion or, alternatively,the lesion is an admixture with a glial neoplasm. Poorly differentiatedneoplasms include, for example, medulloblastomas. Other proliferativedisorders of the CNS, brain or spinal cord include, -primary brainlymphoma, meningiomas, and metastatic tumors.

[0196] It is recognized that administration of the cytokine via themethods of the present invention for the treatment of a proliferativedisorder can be used in combination with any other treatment or therapyknown in the art for the treatment of proliferation disorders. Therapiesused in the treatment of proliferative disorders include, for example,any form of radiation and chemotherapy treatments. See, for example,Hatano et al. (2000) Acta Neurochir 142:633-8, Burton et al. (1999) CurrOpin Oncol. 11:157-61, and Brandes et al. (2000) Anticancer Res20:1913-20; all of which are herein incorporated by reference.

[0197] An “effective amount” of a cytokine for the treatment of aproliferative disease or disorder using the administration method of thepresent invention will be sufficient to reduce or lessen themorphological and/or clinical symptoms of the proliferative disorder. Assuch, an effective amount of the cytokine administered by the methods ofthe present invention will exert any physiological response thatdecreases proliferation of tumor cells and thereby enhances survivaland/or improves clinical status of the treated animal in comparison tothe untreated control. Such physiological responses include, forexample, activation of immune cells, inhibition of cell proliferation,induction of cell differentiation, up-regulation of class I majorhistocompatiblity complex antigens, inhibition of angiogenesis, andestablishment of the T helper 1 (Th1)-type response. Improvement inclinical status includes, for example, an increase in the survival rateof the treated mammal (i.e., an increase in either the one or two yearsurvival rate) and a decrease in tumor size. Assays to measure thesevarious improvements are known in the art. See, for example, Hong et al.(2000) Clin. Cancer Res. 6:3354-60); Knupfer et al. (2000) Cytokine12:409-12; Natsume et al. (1999) Gene Ther 6:1626-33; and U.S. Pat. No.4,846,782, all of which are herein incorporated by reference. Moreextensive details regarding the clinical manifestations of proliferativedisorders of the CNS, brain, spinal cord, or lymphatic system that canbe improved upon the administration of an effective concentration of acytokine can be found in Harrison's Principles of Internal Medicine(McGraw Hill, 1994), herein incorporated by reference.

[0198] The present invention may be better understood with reference tothe following examples. These examples are intended to be representativeof specific embodiments of the invention, and are not intended aslimiting the scope of the invention.

EXPERIMENTAL Example 1

[0199] Intranasal Administration of IFN-β to the CNS

[0200] Introduction

[0201] Administering interferon-P (IFN-β) intranasally is an effectivemeans for delivering this cytokine to the CNS of an animal.

[0202] Materials and Methods

[0203] Intranasal Delivery to the CNS:

[0204] Male Sprague-Dawley rats, 199 and 275 grams, were anesthetizedwith intraperitoneal pentobarbital (40 mg/kg). Drug delivery to the CNSwas assessed after intranasal administration of 51 picomoles and 57picomoles of ¹²⁵I-IFN-β in 20 mM Hepes, pH 7.5, to the light and heavyrat, respectively. Rats were placed on their backs and administered˜100microliters ¹²⁵I-IFN-β to each naris over 10-22 minutes, alternatingdrops every 2-3 minutes between the left and right nares. During theintranasal administration of IFN-β, one side of the nose and the mouthwere held closed. This method of administering the cytokine allows forboth pressure and gravity to deliver the agent into the upper one thirdof the nasal cavity. Rats subsequently underwent perfusion-fixationwithin minutes following the completion of ¹²⁵I-IFN-β administration.Perfusion-fixation was performed with 50-100 ml physiologic salinefollowed by 500 ml of fixative containing 4% paraformaldehyde in 0.1 MSorenson's phosphate buffer, pH 7.4, prior to brain and spinal corddissection and 1251 measurement by gamma counting. Areas dissectedincluded the spinal cord, olfactory bulbs, frontal cortex, anteriorolfactory nucleus, hippocampal formation, choroid plexus, diencephalon,medulla, pons, and cerebellum.

[0205] Results

[0206] Rapid appearance of radiolabel was observed throughout the spinalcord, brain stem, and brain, with the concentrations ranging from about3 pM to about 93 pM. Detailed results are shown below in Table 1. Theobservation of substantial concentrations of interferon-β in theolfactory and trigeminal nerves suggests that this cytokine istransported through or along these nerves. Tissues with biologicallysignificant levels of interferon-P include the olfactory bulbs, frontalcortex, caudate putamen, anterior olfactory nerve, hippocampalformation, choroid plexus, diencephalon, pons, medulla, ventral dura,trigeminal nerve, olfactory epithelium, circle of Willis, and uppercervical spinal cord. TABLE 1 Data for the intranasal (I.N.) delivery ofBetaseron to the CNS Concentration (pM) Concentration (pM) Tissue type(51 picomole dose) (57 picomole dose) Left olfactory bulb 89.5 51.4Right olfactory bulb 92.9 67.7 Frontal cortex 9.19 29.1 Caudate/putamen7.09 34.0 Anterior olfactory nerve 46.9 97.4 Hippocampal formation(left) 5.81 11.7 Hippocampal form (right) 11.1 21.0 Choroid plexus 79.033.2 Diencephalon 15.5 24.0 Midbrain 10.9 19.8 Pons 16.9 49.4 Medulla24.7 90.2 Cerebellum 10.2 30.4 Dura (ventral) 263.0 896 Trigeminal nerve36.7 362 Left olfactory epithelium 3697 Circle of Willis 189 UpperCervical Spinal Cord 24.3 455 Cervical spinal cord 6.88 Thoracic spinalcord 4.0 2.55 Lumbar spinal cord 2.08 3.5 Right olfactory epithelium22,540

[0207] Further quantitation studies for the intranasal delivery of[¹²⁵I]Betaseron were performed in Sprague-Dawley rats essentiallydescribed above. The results are summarized in Table 2. Scans of coronalbrain tissue sections showed prominent labeling of the olfactory bulb,caudate/putamen, septal nucleus, periventricular white matter, opticnerve, and superior colliculus (data not shown). These results are inagreement with the results provided in Table 1. The quantitative studiesperformed in six animals, following internasal administration of about 6nmol of Betaseron, demonstrated consistent delivery to a wide variety ofCNS structures. Highest concentrations of IFN-β were found in theolfactory bulbs (9 nM), anterior olfactory nucleus (3.3 nM), midbrain(1.9 nM), medulla (1.8 nM), pons (1.6 nM), and cerebellum (1.4 nM).Moderate concentrations were observed in the hippocampal formation (1.3nM), diencephalon (1.3 nM), frontal cortex (1.1 nM), cervical spinalcord (1.1 nM), and caudate/putamen (0.83 nM).

[0208] The very high concentrations of [¹²⁵1]Betaseron observed in thetrigeminal nerve (14 nM) and ventral dura mater (19 nM) strongly suggestthat delivery to the CNS involves movement not only along the olfactoryneural pathway but also along the trigeminal nerve pathway. Trigeminaldelivery should result in high levels in both the olfactory areas andmidbrain and brain stem regions. Delivery to the spinal cord probablyoccurs via the trigeminal pathway. Consistent with trigeminal delivery,[¹²⁵I]Betaseron reaches the spinal cord within 25 minutes, and exhibitsdecreasing concentration as you move down the spinal cord.

[0209] These results indicate the direct transport of IFN-β along one ormore neural pathways into the CNS, brain, and spinal cord. TABLE 2Concentration (nM) IFN-β (Betaseron) in Different Rat Tissues FollowingI.N. Administration of ¹²⁵I-IFN-β + IFN-β. Tissue IF11 IF12 IF13 IF14IF15 IF16 Mean SE Blood Sample 1 1.12 1.53 0.92 0.74 1.70 0.85 1.1 0.2Blood Sample 2 2.77 3.26 1.44 2.11 3.13 2.74 2.6 0.3 Blood Sample 3 3.726.62 2.81 3.87 5.02 4.22 4.4 0.5 Blood Sample 4 5.37 6.35 7.38 5.4 7.325.50 6.2 0.4 Blood Sample 5 7.29 6.69 8.15 7.95 7.5 0.3 Left OlfactoryBulb 9.02 6.11 3.01 5.93 18.29 18.46 10 3 Right Olfactory Bulb 5.6 6.993.39 4.84 15.26 12.48 8.1 1.9 Frontal Cortex 1.12 1.24 1.09 0.44 1.721.19 1.1 0.2 Caudate/Putamen 0.68 0.91 0.83 0.36 1.08 1.11 0.83 0.11Ant. Olf. Nucleus 2.11 2.55 1.96 1.09 6.82 5.50 3.3 0.9 L. HippocampalForm. 0.84 1.63 1.24 0.37 2.23 1.71 1.3 0.3 R. Hippocampal Form. 0.851.77 1.24 0.40 1.84 1.91 1.3 0.3 Diencephalon 0.86 1.52 1.39 0.44 2.051.72 1.3 0.2 Midbrain 0.80 1.69 1.53 0.44 5.07 1.91 1.9 0.7 Pons 0.761.91 1.76 0.38 2.71 2.04 1.6 0.4 Medulla 0.63 2.41 2.90 0.42 2.29 2.081.8 0.4 Cerebellum 0.89 1.72 1.56 0.36 2.19 1.84 1.4 0.3 Ventral Dura2.47 46.16 10.89 7.13 21.35 23.52 19 6 Trigeminal Nerve 7.94 12.14 19.894.57 24.44 17.63 14 3 Spinal Dura 0.59 0.13 0.29 0.34 0.13 CervicalSpinal Cord 0.33 0.88 3.12 0.38 0.98 1.00 1.1 0.4 Thoracic Spinal Cord0.14 0.11 0.39 0.29 0.33 0.15 0.24 0.05 Lumbar Spinal Cord 0.13 0.120.27 0.22 0.32 0.10 0.19 0.04 Deltoid Muscle 0.62 0.58 0.50 1.10 0.670.22 0.62 0.12 Liver 0.58 0.78 1.01 1.38 0.54 0.31 0.77 0.16 Kidney 0.670.73 2.08 5.26 0.56 1.81 1.9 0.7 Lung 1.87 0.56 2.18 0.72 0.85 0.99 1.20.3 Esophagus 1.10 1.50 68.2 5234.83 1.44 22.40 888 869 Trachea 1.483.11 83.46 4.67 1.45 5.91 17 13 L. Olfact. Epithelium 1175.9 75.64 14.081431.14 454.41 227.29 563 244 R. Olfact. Epithelium 2083.1 411.32 45.661113.87 191.13 2765.47 1102 453

Example 2

[0210] Intranasal Administration of IFN-β Retains PharmacologicalActivity in the CNS

[0211] Assays were performed to determine if IFN-β, deliveredintranasally, retained pharmacological activity in the CNS. IFN-βactivates signal transduction pathways via a cell surface IFN receptor.The IFN receptor is part of a prototypical JAK-STAT signaling complex.It has two transmembrane chains that associate with intracellularsignaling proteins including TYK2, JAK1, and two latent transcriptionfactors termed “signal transducers and activators of transcription”(STATs). Binding of IFN-β to the receptor brings the two Janus kinases(TYK2 and JAK1) near each other, and they become activated byphosphorylation. The kinases then activate the cytoplasmic tails of theIFN receptors by phosphorylating tyrosine residues. Thesephosphotyrosines provide docking sites for the STATs, bringing them intoappropriate positions for phosphorylation by the nearby Janus kinases.Upon phosphorylation STATs translocate to the nucleus, bind specific DNAelements and direct transcription. Hence, the pharmacological activityof IFN-β following intranasal delivery can be effectively assayed bymonitoring the phosphorylation states of TYK2 and STAT1 throughout thebrain cortex.

[0212] Methods:

[0213] Control/Drug Treatment:

[0214] Harlan Sprague-Dawley rats were anesthetized with pentabarbitol(50 mg/kg). 80 μl of either water or IFN-β was intranasally administeredin 5 doses over a 20 minute time period. Specifically, 8 ,μl wasadministered in 5 doses at 2 minute intervals for each nostril.Recombinant rat interferon-: (rrIFN-β) (35 picomoles) was intranasallyadministered to rat IF35 (drug-treated) and H₂O (vehicle used to diluterrIFN-0) was administered to rat IF33 (control-treated). Afteradministration the animal was perfused with 100 ml of saline and fixedwith 200 ml of 10% formalin. The brain was then removed and sliced in abrain matrix into 2 mm sections. The slices were collected in cassettesand paraffin embedded. Tissue was sliced to 4 μm and placed onmicroscope slides.

[0215] Immunohistological Staining:

[0216] The antibodies to the phosphorylated forms of proteins TYK2 andSTAT1 were purchased from Cell Signaling Technology (product numbers9321L and 9171S, respectively).

[0217] The method of immunohistological staining was as follows. Tissuesections were deparaffinized and hydrated by placing the slides in thefollowing solutions for the indicated times: Xylene for 10 min; 100%EtOH for 5 min; 95% EtOH for 5 min; 70% EtOH for 5 min; and, 50% EtOHfor 5 min. The slides were removed from Coplin jars and washed in H₂Ofor 2 min on a rocking platform. The antigen (TYK2 and/or STAT 1) wasunmasked by incubating slides in citrate buffer (pH 6.0) and heating ina vegetable steamer for 45 min. The slides were removed and washed incold running tap H₂O for 10 min. Slices were incubated in 3% H₂O for 10min at room temperature (RT) in a humid chamber and subsequently washedin H₂O for 5 min. Next, slides were washed in a tris buffered salinesolution (50 mM tris, 150 mm NaCl) with 0.2% Triton X-100 (TBST) forthree 5 min washes. Following the wash, the slides were blocked with 2%goat serum in TBST (GSTBST) for 1 hr at RT. Following three 5 min washesin TBST, the slides were incubated with primary antibody (rabbitanti-TYK2 polycolonal antibody; diluted in GSTBST 1:250) in a humidchamber at RT for 30 min and incubated overnight at 4° C. The next day,the slides were wash in TBST for three 5 min washes and incubated withgoat anti-rabbit secondary antibody. The secondary antibody was diluted1:400 in 10 mM phosphate buffered solution (PBS; 137 mM sodium chloride,2.7 mM potassium chloride) at RT for 1 hr. For the last 15 min of thisincubation, the ABC reagent was made (5 ml PBS, 2 drops of reagent A,mix, 2 drops of reagent B, mix; Vector Technology product # PK-6101) andallowed to stand at RT. Slides underwent an additional three 5 minutewashes in TBST, followed by incubation with ABC reagent at RT for 1 hrin a humid chamber. An additional three 5 minute washes in TBSTfollowed. Approximately 100-150 μl, enough to cover the tissue, ofdiaminobenzidine tetrahydrochloride (DAB) was added and allowed toincubate at RT for 10 min. The reaction was stopped by a 2 minute washwith H₂O. Slides were subsequently washed in H₂O until the solution wasclear. Slides were dehydrated in the following solutions for theindicated times: 50% EtOH for 2 min; 70% EtOH for 2 min; 95% EtOH for 2min; 100% EtOH for 2 min; 50/50 Xylene/ROH for 2 min; and Xylene for 5min. Excess xylene was removed and slides were mounted by adding 2-3drops of Vectamount and covered with coverslip. The Vectamount wasallowed to dry before viewing.

[0218] Results:

[0219] Induction of the IFN-α/β pathway is characterized by thephosphorylation of TYK2 and STAT1. Therefore, antibodies specific to thephosphorylated forms of TYK2 and STAT 1 were used to measure the levelof the activated from of these proteins prior to and followingintranasal delivery of IFN-β. Quantitation revealed that the levels ofphosphorylated TYK2 increased throughout the brain cortex followingintranasal delivery of 35 pmol of recombinant rat IFN-β (data notshown). These results demonstrate that IFN-β retains pharmacologicalactivity in the CNS following the intranasal delivery methods of thepresent invention.

Example 3

[0220] Intranasal Administration of IFN-β to the Lymphatic System

[0221] Intranasal delivery of [125I] Betaseron was performed inSprague-Dawley Rats as essentially described in Example 1. 3.9-7.9 nmolBetaseron was administered in a 44-96 μl volume over the course of 20-29minutes. Animals were perfused at 30 minutes. Data obtained from eightindividual animals is provided in Table 3. Experimental means from thisset of experiments are provided in Table 4. These quantitative studiesdemonstrated delivery of [1251] Betaseron to the superficial cervicalnodes and to the deep cervical nodes of the lymphatic system. Onaverage, 6.1 nM Betaseron was found in the superficial cervical nodes,and 31.5 nM was found in the deep cervical nodes following theadministration methods of the invention. These results are summarized inTable 5. TABLE 3 Betaseron Concentration (nM) Following I.N.Administration of ¹²⁵I-IFNβ + rhIFNβ Experiment IF34 IF36 IF37 IF38MicroCi 31 47 61 48 Nmol 3.9 6.9 7.9 6.6 Blood Sample #1 0.53 0.58 1.11.6 Blood Sample #2 1.6 4.3 2.8 3.9 Blood Sample #3 2.4 3.1 4.6 6.2Blood Sample #4 3.6 4.7 5.1 8.3 Blood Sample #5 4.1 7.0 7.0  10 BloodSample #6 8.2 8.5  10 Left Olfactory Epithelium  65  862  388 643 RightOlfactory Epithelium  62  1103 1447 1876  Left Olfactory Bulb 1.6 3.55.6 Right Olfactory Bulb 1.3 8.1 7.1 Antenor Olfactory Nucleus 0.96 1.32.5 Frontal Cortex 0.28 0.84 0.97 Caudate/Putamen 0.09 0.57 1.7 L + RHippocampus 0.38 0.62 0.82 Left Hippocampus Right HippocampusDiencephalon 0.65 0.74 0.95 Midbrain 0.48 0.61 0.88 Pons 0.45 0.75 0.91Medulla 0.36 0.76 0.95 Cerebellum 0.34 0.54 0.69 Ventral Brain Dura 6.19.7  12  14 Optic Nerve + Chiasm 1.2 6.3 4.4  25 Trigeminal Nerve 5.8  12 8.5  20 Spinal Dura 0.09 0.16 0.67 1.1 Upper Cervical Cord 0.43 2.30.92 1.1 Cervical Spinal Cord 0.17 0.21 0.47 1.3 Thoracic Spinal Cord0.13 0.20 0.35 0.58 Lumbar Spinal Cord 0.17 0.29 0.39 0.49 SuperficialCervical Nodes 8.1 6.3 4.0 6.1 L. Superficial Cervical Node 3.9 R.Superficial Cervical Node 4.2 Deep Cervical Nodes 9.7   16  68 Left DeepCervical Node Right Deep Cervical Node Common Carotids  14   27  38  22Thyroid 250  462  830 725 Esophagus 145  196  394 715 Trachea 177 41863 692 553 Muscle 0.52 0.64 0.74 1.1 Liver 0.47 1.9 0.83 1.2 Kidney 1.00.79 2.92 1.8 Lung 0.66 1.7 2.4  27

[0222] TABLE 4 Experimental Means of Betaseron Concentrations (nM)following I.N. Administration of ¹²⁵I-IFNβ + rhIFNβ Avg for IF34,37,38microCi/nmol 46.67 6.13 Mean Std Dev Blood Sample #1 1.1 0.53 BloodSample #2 2.7 1.2 Blood Sample #3 4.4 1.9 Blood Sample #4 5.7 2.4 BloodSample #5 7.1 3.1 Blood Sample #6 9.5 1.4 Left Olfactory Epithelium 365290 Right Olfactory Epithelium 1128  948 Left Olfactory Bulb 3.6 2.0Right Olfactory Bulb 5.5 3.7 Anterior Olfactory Nucleus 1.6 0.81 FrontalCortex 0.70 0.37 Caudate/Putamen 0.80 0.85 L + R Hippocampus 0.61 0.22Left Hippocampus Right Hippocampus Diencephalon 0.78 0.16 Midbrain 0.660.20 Pons 0.71 0.24 Medulla 0.69 0.30 Cerebellum 0.52 0.17 Ventral BrainDura  11 4.3 Optic Nerve + Chiasm  10  13 Trigeminal Nerve  11 7.5Spinal Dura 0.61 0.49 Upper Cervical Cord 0.83 0.36 Cervical Spinal Cord0.65 0.59 Thoracic Spinal Cord 0.35 0.23 Lumbar Spinal Cord 0.35 0.17Superficial Nodes 6.0 2.1 Left Superficial Cervical Node RightSuperficial Cervical Node Deep Cervical Nodes  39  41 Left Deep CervicalNode Right Deep Cervical Node Common Carotids  25  12 Thyroid 602 309Esophagus 418 286 Trachea 474 266 Muscle 0.78 0.27 Liver 0.82 0.35Kidney 1.9 0.95 Lung  10  15

[0223] TABLE 5 Summary of Betaseron Concentration (nM) in the CervicalLymph Nodes Following I.N. Administration of ¹²⁵I-IFNβ + rhIFNβExperiment IF34 IF36 IF37 IF38 MicroCi 31 47 61 48 Nmol 3.9 6.9 7.9 6.6Average Std Dev Superficial Cervical 8.1 6.3 4.0 6.1 6.1 1.7 Nodes DeepCervical Nodes 9.7 16 68 31 32

Example 4

[0224] Intravenous Administration of Betaseron

[0225] Intravenous administration of Betaseron was studied in order todetermine the extent to which delivery to the CNS and/or lymphaticsystem following intranasal administration may be due to absorption fromthe nasal cavity into the circulation, followed by subsequent deliveryto the CNS and lymphatics.

[0226] Male Harlan Sprague-Dawley rats weighing 263-318 g were used forthese experiments. Rats were anaesthetized with sodium pentobarbital(Nembutal, 50 mg/kg). For each rat, a 500 μl solution containing¹²⁵I-IFN-β and rhIFN-β in 0.9% NaCl was delivered intravenously over60-90 seconds through a cannula into the femoral vein. On average, 560pmol and 49 μCi of IFN-β were administered to each rat. Then 0.2 ml ofblood was collected from the descending aorta cannula every 5 minutesfor a total of five blood samples. Lastly, the rat was perfused throughthe descending aorta cannula with 60-90 ml of 0.9% NaCl followed by 400ml of fixative (4% paraformaldehyde in Sorenson's phosphate buffer).Individual tissue sections were dissected out, placed in 5 ml Startedttubes, and then counted for gamma rays in the Packard Cobra II autogammacounter.

[0227] The methods described above created the same general blood levelof Betaseron with intravenous delivery as that achieved in theintranasal administration studies. Tables 6 and 7 provide the level ofBetaseron in the blood following either intravenous injection andintranasal administration. The level of Betaseron in the blood followingintravenous administration and intranasal administration over time isshown graphically in FIG. 1.

[0228] This study demonstrated that very little of the intravenouslyadministered Betaseron reaches either the CNS or lymphatics.Consequently, it is clear that the intranasal method of deliverydescribed in this application is very beneficial in targeting the CNSand lymphatics of the head and neck region. This method of delivery doesnot utilize the circulation to reach the CNS or lymphatics, but ratherbypasses the circulation and blood-brain barrier to accomplish delivery.Because it is not necessary to use the circulatory system to deliver themedication to the CNS and/or lymphatics, systemic side effects can besignificantly reduced. TABLE 6 Level of Betaseron in the blood followingintravenous administration. Experiment # IF47 IF49 IF50 Mean Std ErrDelivered nmol 0.521 0.579 0.579 0.560 0.019 Delivered uCi 56 46 45 49 4 5 min Blood Sample 6.30 4.47 6.78 5.85 0.70 10 min Blood Sample 5.354.41 5.29 5.01 0.30 15 min Blood Sample 5.90 4.14 5.95 5.33 0.60 20 minBlood Sample 5.95 4.48 5.92 5.45 0.49 25 min Blood Sample 6.40 4.16 6.305.62 0.73

[0229] TABLE 7 Level of Betaseron in the blood stream followingintranasal administration. Experiment # IF36 IF37 IF38 IF40 Mean Std ErrDelivered nmol 6.890 7.947 6.583 7.360 7.195 0.30 Delivered uCi 47 61 4851 52 3  5 min Blood Sample 0.58 1.08 1.60 2.44 1.43 0.40 10 min BloodSample 1.43 2.76 3.89 5.91 3.50 0.95 15 min Blood Sample 3.06 4.62 6.228.30 5.55 1.12 20 min Blood Sample 4.70 5.14 8.27 10.17 7.07 1.30 25 minBlood Sample 6.93 7.04 10.16 12.85 9.25 1.42

[0230] TABLE 8 Concentration (nM) following intravenous administrationof IFN-β. IF47 IF49 IF50 Mean Std Err Delivered nmol 0.521 0.579 0.5790.560 0.019 Delivered uCi 56 46 45 49 4 Blood Sample #1 (5 min) 6.304.47 6.78 5.85 0.70 Blood Sample #2 (10 min) 5.35 4.41 5.29 5.02 0.30Blood Sample #3 (15 min) 5.90 4.14 5.95 5.33 0.60 Blood Sample #4 (20min) 5.95 4.47 5.92 5.45 0.49 Blood Sample #5 (25 min) 6.40 4.16 6.305.62 0.73 Left Olfactory Epithelium 0.27 0.72 0.86 0.62 0.18 RightOlfactory Epithelium 0.19 0.78 1.04 0.67 0.25 Left Olfactory Bulb 0.630.23 0.31 0.39 0.12 Right Olfactory Bulb 1.01 0.22 0.23 0.49 0.26Anterior Olfactory Nucleus 0.15 0.13 0.17 0.15 0.01 Frontal Cortex 0.150.16 0.18 0.16 0.01 Caudate/Putamen 0.21 0.11 0.15 0.16 0.03 Hippocampus0.13 0.11 0.14 0.13 0.01 Cerebellum 0.15 0.12 0.16 0.14 0.01Diencephalon 0.14 0.12 0.14 0.13 0.01 Midbrain 0.16 0.12 0.14 0.14 0.01Pons 0.13 0.11 0.03 0.09 0.03 Medulla 0.14 0.11 0.14 0.13 0.01 DorsalBrain Dura 0.41 0.43 0.42 0.01 Ventral Brain Dura 1.32 0.28 0.17 0.590.37 Optic Nerve + Chiasm 0.18 0.29 0.24 0.04 Trigeminal Nerve 0.28 0.210.26 0.25 0.02 Spinal Dura 0.07 0.13 0.12 0.11 0.02 Upper Cervical Cord0.15 0.12 0.09 0.12 0.02 Cervical Cord 0.10 0.10 0.09 0.10 0.00 ThoracicSpinal Cord 0.08 0.09 0.11 0.09 0.01 Lumbar Spinal Cord 0.11 0.12 0.140.12 0.01 Superficial Nodes 0.42 0.28 0.64 0.45 0.10 Deep Cervical Nodes0.10 0.34 0.40 0.28 0.09 Axial Nodes 0.33 0.64 0.49 0.13 Common Carotids0.13 0.11 0.09 0.11 0.01 Thyroid 52.65 56.49 11.03 40.06 14.56 Esophagus0.92 0.91 0.29 0.71 0.21 Trachea 0.81 0.49 0.46 0.59 0.11 Deltoid Muscle0.29 0.19 0.30 0.26 0.04 Liver 15.17 11.82 16.90 14.63 1.49 Kidney 1.301.51 1.49 1.43 0.07 Lung 16.02 30.02 33.14 26.39 5.26

[0231] It should be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to a composition containing “a compound” includes amixture of two or more compounds.

[0232] All publications and patent applications in this specificationare indicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

[0233] The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

That which is claimed:
 1. A method for transporting a cytokine to acentral nervous system of a mammal, comprising: administering acomposition comprising the cytokine to a tissue of the mammal innervatedby the trigeminal nerve, the olfactory nerve, or a combination thereof,wherein the cytokine is absorbed through the tissue and transported tothe central nervous system of the mammal.
 2. The method of claim 1 ,wherein the tissue comprises a nasal cavity tissue, a conjunctiva, anoral tissue, or a skin.
 3. The method of claim 2 , wherein administeringthe cytokine to the conjunctiva comprises administering the cytokinebetween a lower eyelid and an eye.
 4. The method of claim 2 , whereinadministering the cytokine to the skin comprises administering thecytokine to a face, a forehead, an upper eyelid, a lower eyelid, adorsum of the nose, a side of the nose, an upper lip, a cheek, a chin, ascalp, or a combination thereof.
 5. The method of claim 2 , whereinadministering the cytokine to the oral tissue comprises sublingualadministration.
 6. The method of claim 1 , wherein said cytokine isselected from the group consisting of interferon-alpha (IFN-α),interferon-beta (IFN-β), interferon-gamma (IFN-γ), and biologicallyactive variants thereof.
 7. The method of claim 6 , wherein the IFN-β ishuman IFN-β or a biologically active variant thereof.
 8. The method ofclaim 7 , wherein the IFN-β is biologically active and comprises anamino acid sequence having at least 70% sequence identity to humanIFN-β.
 9. The method of claim 1 , wherein the cytokine is administeredto an upper one third of a nasal cavity.
 10. The method of claim 1 ,wherein the cytokine is transported to a cerebellum, a superiorcolliculus, a periventricular white matter, an optic nerve, a midbrain,a pons, an olfactory bulb, an anterior olfactory nucleus, or anycombination thereof.
 11. The method of claim 1 , wherein the cytokine istransported to a spinal cord, a brain stem, a cortical structure, asubcortical structure, or any combination thereof.
 12. The method ofclaim 1 , wherein the cytokine is administered in a dosage range ofabout 0.14 nmol/kg of brain weight to about 138 nmol/kg of brain weight.13. The method of claim 12 , wherein the cytokine is human IFN-β or abiologically active variant thereof.
 14. A method for administering acytokine to a central nervous system of a mammal, comprising:administering a composition comprising an effective amount of thecytokine to a tissue of the mammal innervated by the trigeminal nerve,the olfactory nerve, or a combination thereof, wherein the cytokine isabsorbed through the tissue and transported into the central nervoussystem of the mammal in an amount effective to provide a diagnostic,protective, or therapeutic effect on a cell of the central nervoussystem.
 15. The method of claim 14 , wherein the tissue comprises atissue of a nasal cavity, a conjunctiva, an oral tissue, or a skin. 16.The method of claim 15 , wherein administering the cytokine to theconjunctiva comprises administering the cytokine between a lower eyelidand an eye.
 17. The method of claim 15 , wherein administering thecytokine to the skin comprises administering the cytokine to a face, aforehead, an upper eyelid, a lower eyelid, a dorsum of the nose, a sideof the nose, an upper lip, a cheek, a chin, a scalp, or a combinationthereof.
 18. The method of claim 15 , wherein administering the cytokineto the oral tissue comprises sublingual administration.
 19. The methodof claim 14 , wherein the cytokine is transported to lymphaticsassociated with the central nervous system.
 20. The method of claim 14 ,wherein said cytokine is selected from the group consisting of IFN-α,IFN-β, IFN-γ, and biologically active variants thereof.
 21. The methodof claim 20 , wherein said IFN-β is human IFN-β or a biologically activevariant thereof.
 22. The method of claim 21 , wherein said IFN-β orvariant thereof retains biological activity and comprises an amino acidsequence having at least 70% sequence identity to the sequence of humanIFN-β.
 23. The method of claim 14 , wherein the cytokine is delivered toan upper one third of a nasal cavity.
 24. The method of claim 14 ,wherein the cytokine is transported to the central nervous system of themammal in an amount effect for preventing or reducing a viral infection.25. The method of claim 24 , wherein said viral infection is selectedfrom the group consisting of viral meningitis, herpes simplex, hepatitisC, and human immunodeficiency (HIV).
 26. The method of claim 14 ,wherein the cytokine is transported to the central nervous system of themammal in an amount effective to treat or prevent a disordercharacterized by an immune or an inflammatory response.
 27. The methodof claim 26 , wherein said disorder is selected from the groupconsisting of Alzheimer's disease, meningitis, Primary Sjogren'sSyndrome, multiple sclerosis, and HIV.
 28. The method of claim 14 ,wherein the cytokine is transported to the central nervous system of themammal in an amount effective to treat or prevent a proliferativedisorder.
 29. The method of claim 28 , wherein said proliferativedisorder is a glioma.
 30. The method of claim 14 , wherein the cytokineis administered in a dosage range from about 0.14 nmol/kg of brainweight to about 138 nmol/kg of brain weight.
 31. The method of claim 30, wherein the cytokine is human IFN-β or a biologically active variantthereof.
 32. A method for transporting a cytokine to a lymphatic systemof a mammal, comprising: administering a composition comprising thecytokine to a tissue of the mammal innervated by the trigeminal nerve,the olfactory nerve, or a combination thereof, wherein the cytokine isabsorbed through the tissue and transported to the lymphatic system ofthe mammal.
 33. The method of claim 32 , wherein the tissue comprises anasal cavity tissue, a conjunctiva, an oral tissue, or a skin.
 34. Themethod of claim 33 , wherein administering the cytokine to theconjunctiva comprises administering the cytokine between a lower eyelidand an eye.
 35. The method of claim 33 , wherein administering thecytokine to the skin comprises administering the cytokine to a face, aforehead, an upper eyelid, a lower eyelid, a dorsum of the nose, a sideof the nose, an upper lip, a cheek, a chin, a scalp, or a combinationthereof.
 36. The method of claim 33 , wherein administering the cytokineto the oral tissue comprises sublingual administration.
 37. The methodof claim 32 , wherein said cytokine is selected from the groupconsisting of interferon-alpha (IFN-α), interferon-beta (IFN-β),interferon-gamma (IFN-γ), and biologically active variants thereof. 38.The method of claim 37 , wherein the IFN-β is human IFN-β or abiologically active variant thereof.
 39. The method of claim 38 ,wherein the IFN-β is biologically active and comprises an amino acidsequence having at least 70% sequence identity to human IFN-β.
 40. Themethod of claim 32 , wherein the cytokine is administered to an upperone third of a nasal cavity.
 41. The method of claim 32 , wherein thecytokine is transported to a deep cervical node, a superficial cervicalnode, or a combination thereof.
 42. The method of claim 32 , wherein thecytokine is administered in a dosage range of about 0.14 nmol/kg ofbrain weight to about 138 nmol/kg of brain weight.
 43. The method ofclaim 42 , wherein the cytokine is human IFN-β or a biologically activevariant thereof.
 44. A method for administering a cytokine to alymphatic system of a mammal, comprising: administering a compositioncomprising an effective amount of the cytokine to a tissue of the mammalinnervated by the trigeminal nerve, the olfactory nerve, or acombination thereof, wherein the cytokine is absorbed through the tissueand transported into the lymphatic system of the mammal in an amounteffective to modulate an immune or inflammatory response.
 45. The methodof claim 44 , wherein the tissue comprises a tissue of a nasal cavity, aconjunctiva, an oral tissue, or a skin.
 46. The method of claim 45 ,wherein administering the cytokine to the conjunctiva comprisesadministering the cytokine between a lower eyelid and an eye.
 47. Themethod of claim 45 , wherein administering the cytokine to the skincomprises administering the cytokine to a face, a forehead, an uppereyelid, a lower eyelid, a dorsum of the nose, a side of the nose, anupper lip, a cheek, a chin, a scalp, or a combination thereof.
 48. Themethod of claim 45 , wherein administering the cytokine to the oraltissue comprises sublingual administration.
 49. The method of claim 44 ,wherein said cytokine is selected from the group consisting of IFN-α,IFN-β, IFN-γ, and biologically active variants thereof.
 50. The methodof claim 49 , wherein said IFN-β is human IFN-β or a biologically activevariant thereof.
 51. The method of claim 50 , wherein said IFN-β orvariant thereof retains biological activity and comprises an amino acidsequence having at least 70% sequence identity to the sequence of humanIFN-β.
 52. The method of claim 44 , wherein the cytokine is delivered toan upper one third of a nasal cavity.
 53. The method of claim 44 ,wherein the cytokine is administered in a dosage range of about 0.14nmol/kg of brain weight to about 138 nmol/kg of brain weight.
 54. Themethod of claim 53 , wherein the cytokine is human IFN-β or abiologically active variant thereof.
 55. The method of claim 44 ,wherein the cytokine is transported to the lymphatic system of themammal in an amount effect for preventing or reducing a viral infection.56. The method of claim 55 , wherein said viral infection is selectedfrom the group consisting of viral meningitis, herpes simplex, hepatitisC, and human immunodeficiency (HIV).
 57. The method of claim 44 ,wherein the cytokine is transported to the lymphatic system of themammal in an amount effective to treat or prevent a disordercharacterized by an immune or an inflammatory response.
 58. The methodof claim 57 , wherein said disorder is selected from the groupconsisting of Alzheimer's disease, meningitis, Primary Sjogren'sSyndrome, multiple sclerosis, and HIV.
 59. The method of claim 44 ,wherein the cytokine is transported to the lymphatic system of themammal in an amount effective to treat or prevent a proliferativedisorder.
 60. The method of claim 59 , wherein said proliferativedisorder is a glioma.